Compositions and methods to control insect pests

ABSTRACT

Methods and compositions are provided which employ a silencing element that, when ingested by a plant insect pest, such as Coleopteran, Hemiptera, or Lepidopteran plant pest, including a Diabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan, Bemisia, Halyomorpha, Nezara, or Spodoptera plant pest, decrease the expression of a target sequence in the pest. Disclosed are various target polynucleotides set forth in any one of SEQ ID NOS: 1-53 or 107-254 disclosed herein, or variants and fragments thereof, or complements thereof, wherein a decrease in expression of one or more of the sequences in the target pest controls the pest and pest population by insect sterilization (i.e., male and female sterility, reduction of sperm count, egg production and viability) and mating-based sterile insect technique (SIT). Plants, plant parts, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application NumberPCT/US2016/037748 filed Jun. 16, 2016, which claims the benefit of U.S.Provisional Application No. 62/180,504, filed Jun. 16, 2015, and U.S.Provisional Application No. 62/272,994, filed Dec. 30, 2015, which arehereby incorporated herein in its entirety by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A sequence listing having the file name “6030WOPCT_SequenceList.txt”created on May 19, 2016 and having a size of 531 kilobytes is filed incomputer readable form concurrently with the specification. The sequencelisting is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of molecular biologyand gene silencing to control pests.

BACKGROUND

Plant insect pests are a serious problem in agriculture. They destroymillions of acres of staple crops such as corn, soybeans, peas, andcotton. Yearly, plant insect pests cause over $100 billion dollars incrop damage in the U.S. alone. In an ongoing seasonal battle, farmersmust apply billions of gallons of synthetic pesticides to combat thesepests. Other methods employed in the past delivered insecticidalactivity by microorganisms or genes derived from microorganismsexpressed in transgenic plants. For example, certain species ofmicroorganisms of the genus Bacillus are known to possess pesticidalactivity against a broad range of insect pests including Lepidoptera,Diptera, Coleoptera, Hemiptera, and others. In fact, microbialpesticides, particularly those obtained from Bacillus strains, haveplayed an important role in agriculture as alternatives to chemical pestcontrol. Agricultural scientists have developed crop plants withenhanced insect resistance by genetically engineering crop plants toproduce insecticidal proteins from Bacillus. For example, corn andcotton plants genetically engineered to produce Cry toxins (see, e.g.,Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998)Microbiol. Mol. Biol. Rev. 62(3):775-806) are now widely used inagriculture and have provided the farmer with an alternative totraditional insect-control methods. However, in some instances these Btinsecticidal proteins may only protect plants from a relatively narrowrange of pests. Evolving insect resistance has also presented an issue(Gassmann et al. (2014) PNAS 111(14):5141-6). Thus, novel insect controlcompositions remain desirable.

BRIEF SUMMARY

Methods and compositions are provided which employ a silencing elementthat, when ingested by a plant insect pest, such as Coleopteran,Hemiptera, or Lepidopteran plant pest, including a Diabrotica,Leptinotarsa, Phyllotreta, Acyrthosiphan, Bemisia, Halyomorpha, Nezara,or Spodoptera plant pest, is capable of decreasing the expression of atarget sequence in the pest. In certain embodiments, the decrease inexpression of the target sequence controls the ability of the pest toreproduce, and thereby the methods and compositions are capable oflimiting damage to a plant or the spread of insect pests. Describedherein are various target polynucleotides as set forth in SEQ ID NOS.:1-53 or 107-254, or variants or fragments thereof, or complementsthereof, that modulate the expression of one or more of the sequences inthe target pest RNAs involved in pest reproduction and fecundity.Accordingly, the various target polynucleotides as set forth in SEQ IDNOS.: 1-53 or 107-254, or variants or fragments thereof, or complementsthereof, are useful in methods described herein to control target pestsby insect sterilization and release of sterile target pests, i.e.,sterile insect technique (“SIT”). Also provided are silencing elements,which when ingested by the pest, decrease the level of expression of oneor more of the target polynucleotides. Further provided are constructsencoding silencing elements and host cells comprising constructsencoding silencing elements. Plants, plant parts, plant cells, bacteriaand other host cells comprising the silencing elements or an activevariant or fragment thereof are also provided. Also provided areformulations of sprayable silencing agents for topical applications topest insects or substrates where pest insects may be found.

In another embodiment, a method for controlling a plant insect pest,such as a Coleopteran, Hemiptera, or Lepidopteran plant pest, includinga Diabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan, Bemisia,Halyomorpha, Nezara, or Spodoptera plant pest, is provided. The methodcomprises feeding to a plant insect pest a composition comprising asilencing element, wherein the silencing element, when ingested by thepest, reduces the level of a target sequence in the pest and therebycontrols the pest. Further provided are methods to protect a plant froma plant insect pest. Such methods comprise introducing into the plant orplant part a disclosed silencing element. When the plant expressing thesilencing element is ingested by the pest, the level of the targetsequence is decreased and the pest is controlled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show representative data pertaining to sterilization ofadult Western Corn Rootworm (“WCRW”) following ingestion of anartificial diet comprising a dsRNA construct comprising a targetnucleotide sequence of SEQ ID NO.: 4. FIG. 1A shows the total number ofeggs produced within 13-14 days by treatment and age group. For theyounger female group, 50 pairs of male and female beetles were used, andfor the older female group 50 mated female beetles were used. FIG. 1Bshows the average number of eggs produced per female/day during 13-14day oviposition period by treatment and age group. The box plot graph isproduced by Spotfire program indicating 4 quartiles, average, and 95%confidence interval of the mean. FIG. 1C shows the effect of varioustreatments, as indicated in the figure, on overall average egg hatchrate. Data represents 13-14 days egg collection period; n=6replication/treatment/day; 5-45 eggs/replication depending on the day(p<0.001). FIG. 1D shows gene suppression analysis in WCRW adult beetles8 days after treatment of female and male insects for younger age groupand 4 days after treatment of female insects for older age group.Relative expression of VgR is shown from 4 individual insects for eachtreatment using the DV-RPS10 gene as reference and untreated olderbeetle as normalizer. Box plot shows 4 quartiles, average, median, and95% confidence interval of the mean by treatment and age group.

FIGS. 2A-2B show representative data pertaining to sterilization of WCRWfollowing feeding 3^(rd) instar larvae with an artificial dietcomprising a dsRNA construct comprising a target nucleotide sequence ofSEQ ID NO.: 4. FIG. 2A shows the average numbers and viable eggsproduced per female. Eggs from 15-42 female adult beetles were countedfor each treatment. The number in the box shows average numbers of eggsor viable eggs/female. The box plot shows 4 quartiles, average, median,and 95% confidence interval of the mean for each treatment. For the VgRdsRNA exposed group, viable egg production remain very low throughoutthe study period. Treatment with VgR dsRNA did not affect adultemergence. Mortality of adult beetles due to VgR dsRNA larval exposurewas negligible. FIG. 2B shows VgR gene suppression analysis in 4 10-dayold beetles and more than 15 28-day old beetles at Days 40 and 58 aftertreatment, respectively. Box plot of relative expression by qRTPCR shows4 quartiles, average, median, and 95% confidence interval of the meanfor each treatment in 10 and 28 day old beetles. Untreated 3^(rd) instarlarvae were used as normalizer.

FIGS. 3A-3C show data pertaining to the dose response of WCRWsterilization and gene suppression in WCRW following exposure to anartificial diet comprising a dsRNA comprising a target nucleotidesequence of SEQ ID NO.: 3. FIG. 3A shows the total number of eggs andeggs/female produced during 18 days study period in response to VgRdsRNA doses. Eggs were collected and counted over 18 day ovipositionperiod. Viable eggs/female and net reduction in fecundity (%) areindicated in the last two columns. Net reduction in fecundity (NRF) ofVgR dsRNA treated females relative to control (water exposed females)was estimated using the formula described in the Examples. FIG. 3B showsa box plot of percentage of overall egg hatch rates by dose. Datarepresents 18 days egg collection period; n=1-4replication/treatment/day; 5-478 eggs/replication depending on the dayand availability of eggs. FIG. 3C shows a box plot of relativeexpression of VgR Day 6 after dsVgR treatment at different doses.Untreated beetles were used as normalizer.

FIGS. 4A-4B show data pertaining to VgR gene suppression followingingestion of various VgR dsRNA fragments. FIG. 4A shows schematicdepiction of the VgR fragments and amplicons of qRTPCR assays (indicatedby dashed circles) on VgR coding DNA sequence (“CDS”). FIG. 4B shows abox plot of relative VgR expression 6 days after treatment with dsVgRfragments and controls (ddH2O and dsGUS) using 5′-qRTPCR assay. 4quartiles, average (horizontal solid line), median (horizontal dashline), and 95% confidence interval of the mean are shown. Similarresults were also obtained with Mid- and 3′-qRTPCR assays. Data werenormalized to results obtained from untreated 3rd instar larvae.

FIGS. 5A-5D show data pertaining to VgR fragment screen using genesuppression analysis. FIG. 5A shows a schematic depiction of the VgRfragments used in screen for gene suppression analysis. FIGS. 5B-5Dshows representative gene analysis for the indicated VgR fragments usingresults obtained in three experiments. In each experiment, treatments bywater, GUS, and VgR fragment 1 (SEQ ID NO.:3) were included as controls.Data were normalized to beetles treated with water. Two qRTPCR assays(5′- and Mid-qRTPCR assays) were used to avoid overlapping of VgRfragment and PCR amplicon.

FIGS. 6A-6B show data pertaining to VgR gene suppression in beetlesingesting transgenic plants expressing VgR dsRNA constructs as indicatedin the figure. VgR expression in planta is indicated at the bottom ofeach figure. FIG. 6A shows data in plants at about the V4 growth stagewhich were infested with at least 14 young female beetles in cages. Theplant type is as indicated in the figure, with “NTG” indicatingnon-transgenic control plants; “Frag1” indicates transgenic plantsexpressing a silencing element comprising VgR-Frag1 (SEQ ID NO.: 3);“Frag2” indicates transgenic plants expressing a silencing elementcomprising VgR-Frag2 (SEQ ID NO.: 4), and “Frag3” indicates transgenicplants expressing a silencing element comprising VgR-Frag3 (SEQ ID NO.:5), Beetles were collected 8 days after feeding for gene suppressionanalysis. Data were normalized to data from beetles ingesting the NTGcontrol. FIG. 6B shows data obtained from individual R1 maize plantswere infested with more than 6 young female beetles in cages. Beetleswere collected 12 days after feeding. Each fragment and control isrepresented by 2 plants used for feeding and more than 12 insects usedin gene suppression analysis.

FIG. 7 shows data pertaining to a fecundity assessment of VgR T1 adultbeetle exposure bioassay. For each construct 2-4 events were tested.Each cage received an oviposition dish daily and/or at interval of 2-4days and eggs were subsequently processed.

FIG. 8 shows data pertaining to fecundity assessment of VgR T1 larvalexposure bioassay. For each event three replicate cages containing atleast 8-14 pairs of male and female beetles were arranged. Each cagereceived oviposition dish every 5 days, and eggs were processed

FIGS. 9A-9B show data pertaining to WCRW adult sterilization bioassayand gene suppression by DV-BOULE-FRAG1 (SEQ ID NO: 164) dsRNA treatment.FIG. 9A shows the total number of eggs and fertile eggs produced perfemale; average egg hatch rate with standard error of the mean;reduction in total egg production per female and net reduction infecundity of female beetles relative to water control. FIG. 9B showsgene expression in beetles after BOULE dsRNA treatment. Relativeexpression by qRTPCR assay was described in previous examples. The boxplot shows four quartiles, average (horizontal dash line), median(horizontal solid line), and 95% confidence interval of the mean areshown.

FIG. 10 shows data pertaining to beetle counts from larval exposure toBOULE FRAG1 (SEQ ID NO: 164) dsRNA-expressing T1 transgenic plants. Thebox plot shows four quartiles, average (horizontal dash line), median(horizontal solid line), and 95% confidence interval of the mean.Average expression levels of the BOULE dsRNA fragment in planta for eachevent were determined in root samples using in vitro transcription (IVT)product as control.

FIGS. 11A-11C show data pertaining to WCRW larval exposure to BOULEtransgenic T1 plants causing adult sterilization. FIG. 11A shows theeffect of larval exposure to transgenic plants (expressingDV-BOULE-FRAG1, SEQ ID NO: 164) on the overall average egg productionper female and average viable eggs produced per female from emergedbeetles. Line in each bar represents the standard error of the mean(±SEM) and the same color bars followed by the same upper or lower caseletters are not statistically different. FIG. 11B shows the effect oflarval exposure to transgenic plants (expressing DV-BOULE-FRAG1, SEQ IDNO: 164) on hatch rate of eggs obtained from the emerged beetles. Thebox plot shows four quartiles, average (horizontal white line) and 95%confidence interval of the mean (vertical black line). The average andthe corresponding standard error of the means ((±SEM) are indicated atthe bottom of the box plot. For each treatment egg hatch test wasperformed for 5 batches of eggs and a total of at least 1200-1285 eggsper treatment were assessed for viability. FIG. 11C indicates the effectof larval exposure to transgenic plants (expressing DV-BOULE-FRAG1, SEQID NO: 164) on net reduction in fecundity of emerged adult beetlesrelative to NTG control. The box plot shows four quartiles, average(horizontal black line) and 95% confidence interval of the mean(vertical black line). The average and the corresponding standard errorof the mean are indicated at the bottom of the box plot.

FIG. 12 shows data pertaining to 3rd instar sterilization bioassay ofdsRNA targeting DV-CUL3-FRAG1, DV-NCLB-FRAG1, and DV-MAEL-FRAG1 dsRNA(SEQ ID No.: 44, 45, and 46 respectively) at 1 ppm. The average totalnumber of eggs produced per female, the average number of viable eggsproduced per female, the average egg hatch rate; average reduction inegg production and net reduction in fecundity (both relative to watercontrol) are shown. For each parameter the respective standard error ofthe mean are presented.

DETAILED DESCRIPTION

The disclosures herein will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allpossible embodiments are shown. Indeed, disclosures may be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will satisfy applicable legal requirements.

Many modifications and other embodiments disclosed herein will come tomind to one skilled in the art to which the disclosed compositions andmethods pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the disclosures are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting. As used in the specification and in the claims, the term“comprising” can include the aspect of “consisting of.” Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the disclosed compositions and methods belong. In thisspecification and in the claims which follow, reference will be made toa number of terms which shall be defined herein.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

I. Overview

Methods and compositions are provided which employ one or more silencingelements that, when ingested by a plant insect pest, such asColeopteran, Hemiptera, or Lepidopteran plant pest, including aDiabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan, Bemisia,Halyomorpha, Nezara, or Spodoptera plant pest, are capable of decreasingthe expression of a target sequence in the pest. In certain embodiments,the decrease in expression of the target sequence controls the abilityof the pest to reproduce, and thereby the methods and compositions arecapable of limiting damage to a plant or the spread of insect pests.Disclosed herein are target polynucleotides as set forth in SEQ ID NOS.:1-53 or 107-254, or variants and fragments thereof, and complementsthereof. Silencing elements comprising sequences, complementarysequences, active fragments or variants of these target polynucleotidesare provided which, when ingested by or when contacting the pest,decrease the expression of one or more of the target sequences andthereby controls the pest population via, for example, insectsterilization or through the application of sterile insect technique(SIT; i.e., the silencing elements are associated with sterilizationactivity). In some embodiments, a transgenic plant comprising apolynucleotide encoding silencing elements are provided which, wheningested by or when contacting the pest, decrease the expression of oneor more of the target sequences and thereby controls the pest populationvia, for example, insect sterilization or through the application ofSIT.

In one embodiment, a method relates to producing sterile insects;releasing sterile insects into the environment in very large numbers(about 10 to 100 times the number of native insects) in order to matewith the native insects that are present in the environment, wherein thenative female that mates with a sterile male produce infertile eggs. Ina further embodiment, releasing sterile insects is repeated one or moretimes, wherein the number of native insects decreases and the ratio ofsterile to native insects increases, driving the native population sizedownwards.

It is understood that target pest RNAs can be involved in one or more ofmale and/or female sterility, reduction of sperm count, egg production(fecundity), gender ratios, rates of fertilization (fertility),maturation of sexual organs, and sperm or egg viability. SIT has beenused to control insect population by mating-based approach throughrelease of sterile insects of one or both genders. In one embodiment,SIT comprises release of large number of sterile male insects thatsearch for and mate with wild females, thereby preventing offspring. SITusing different schemes to generate sterile insects has been reported tocontrol mosquito populations such as Anopheles or Aedes (e.g., seeWhyard, et al. (2015) Parasit. Vectors, 8:96; Benedict, M. Q. and A. S.Robinson (2003) Trends Parasitol. 19(8):349; and Nolan, et al. (2011)Genetica 139:33). SIT has been field evaluated for population control ofAedes aegypti in Brazil (Carvalho, D. O. (2015), PLoS. 9(7): e0003864).Dengue, chikungunya, and now Zika virus are all transmitted by Aedesaegypti, one of the most widespread disease-carrying vectors on theglobe.

In one embodiment, a method relates to producing sterile insects;releasing sterile insects into an environment in about 0.5, 1, 5, 10,20, 30, 50, 60, 70, 90, to 100 times the number of native insects,wherein the sterile insects mate with the native insects that arepresent in the environment, and wherein the native female that mateswith a sterile male produce infertile eggs. In a further embodiment,releasing sterile insects is repeated one or more times, wherein thenumber of native insects decreases and the ratio of sterile to nativeinsects increases, driving the native population size downwards.

In one embodiment, compositions and methods are provided which employ aribonucleic acid construct comprising at least one double-stranded RNAregion, at least one strand of which comprises a polynucleotide that iscomplementary to: (a) a nucleotide sequence comprising a sequence of anRNA transcript expressed in a target pest, wherein the down-regulationof the RNA transcript results in increased sterility in the target; orvariants and fragments thereof, and complements of said nucleotidesequence; (b) the nucleotide sequence comprising at least 90% sequenceidentity to said nucleotide sequence; or variants and fragments thereof,and complements thereof; or (c) the nucleotide sequence comprising atleast 19 consecutive nucleotides of said nucleotide sequence; orvariants and fragments thereof, and complements thereof; wherein thepolynucleotide encodes a silencing element having sterilization activityagainst an insect plant pest.

In further embodiment, compositions and methods are provided whichemploy a ribonucleic acid construct comprising at least onedouble-stranded RNA region, at least one strand of which comprises apolynucleotide that is complementary to: (a) a nucleotide sequencecomprising a sequence of an RNA transcript expressed in a Coleopteranpest, wherein the down-regulation of the RNA transcript results inincreased sterility in the target; or variants and fragments thereof,and complements of said nucleotide sequence; (b) the nucleotide sequencecomprising at least 90% sequence identity to said nucleotide sequence;or variants and fragments thereof, and complements thereof; or (c) thenucleotide sequence comprising at least 19 consecutive nucleotides ofsaid nucleotide sequence; or variants and fragments thereof, andcomplements thereof; wherein the polynucleotide encodes a silencingelement having sterilization activity against an insect plant pest.

In another embodiment, compositions and methods are provided whichemploy a ribonucleic acid construct comprising at least onedouble-stranded RNA region, at least one strand of which comprises apolynucleotide that is complementary to: (a) the nucleotide sequencecomprising any one of SEQ ID NOS: 1-53 or 107-254; or variants andfragments thereof, and complements thereof; (b) the nucleotide sequencecomprising at least 90% sequence identity to any one of nucleotides SEQID NOS: 1-53 or 107-254; or variants and fragments thereof, andcomplements thereof; or (c) the nucleotide sequence comprising at least19 consecutive nucleotides of any one of SEQ ID NOS: 1-53 or 107-254; orvariants and fragments thereof, and complements thereof; wherein thepolynucleotide encodes a silencing element having sterilization activityagainst an insect plant pest.

As used herein, “VgR protein” or “vitellogenin receptor protein” refersto a family of large (180-214 kDa), membrane-bound proteins, and includeproteins such as the VgR protein having the sequence of SEQ ID NO.: 106,and variants, homologs, and mutants thereof. It is believed that theseproteins bind with high affinity to vitellogenin (K_(d) values of about30-180 nM) and are involved in the cellular uptake of vitellogenin. VgRprotein is typically expressed in ovarian tissue. As used herein,“BOULE” refers to a family of genes that encode a RNA binding proteinwith a highly conserved RRM (RNA recognition motif) domain and at leastone DAZ (deleted in azoospermia) repeat of 24 amino acids rich in Asn,Tyr, and Gln residues. Deletion or mutations of BOULE in fly usuallyseverely impair spermatogenesis. BOULE is required for meiotic entry andgermline differentiation at the transition between G2 and M phases ofmeiosis. BOULE is typically expressed in germline cells.

As used herein, “VgR mRNA” or “vitellogenin receptor mRNA” refers to amessenger RNA transcript that when translated provides a VgR protein, ora variant, homolog, or mutant protein thereof.

As used herein, by “controlling a plant insect pest” or “controls aplant insect pest” is intended any effect on a plant insect pest thatresults in limiting the damage that the pest causes. Controlling a plantinsect pest includes, but is not limited to, killing the pest,inhibiting development of the pest, altering fertility or growth of thepest in such a manner that the pest provides less damage to the plant,or in a manner for decreasing the number of offspring produced,producing less fit pests, including offspring, producing pests moresusceptible to predator attack, producing pests more susceptible toother insecticidal proteins, or deterring the pests from eating theplant.

Reducing the level of expression of the target polynucleotide or thepolypeptide encoded thereby, in the pest results in the suppression,control, and/or killing the invading pest. In one embodiment, reducingthe level of expression of the target sequence of the pest will reducethe pest damage by at least about 2% to at least about 6%, at leastabout 5% to about 50%, at least about 10% to about 60%, at least about30% to about 70%, at least about 40% to about 80%, or at least about 50%to about 90% or greater. Hence, methods disclosed herein can be utilizedto control pests, including but not limited to, Coleopteran plant insectpests or a Diabrotica plant pest.

Certain assays measuring the control of a plant insect pest are commonlyknown in the art, as are methods to record nodal injury score. See, forexample, Oleson et al. (2005) J. Econ. Entomol. 98:1-8. Other assaymethods are provided in the examples below.

Disclosed herein are compositions and methods for protecting plants froma plant insect pest, or inducing resistance in a plant to a plant insectpest, such as Coleopteran plant pests or Diabrotica plant pests or otherplant insect pests. Plant insect pests include insects selected from theorders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera,Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera andColeoptera.

Those skilled in the art will recognize that not all compositions areequally effective against all pests. Disclosed compositions, includingthe silencing elements disclosed herein, display activity against plantinsect pests, which may include economically important agronomic,forest, greenhouse, nursery ornamentals, food and fiber, public andanimal health, domestic and commercial structure, household and storedproduct pests.

As used herein “Coleopteran plant pest” is used to refer to any memberof the Coleoptera order. Other plant insect pests that may be targetedby the methods and compositions disclosed herein, but are not limited toMexican Bean Beetle (Epilachna varivestis), and Colorado potato beetle(Leptinotarsa decemlineata).

As used herein, the term “Diabrotica plant pest” is used to refer to anymember of the Diabrotica genus. Accordingly, the compositions andmethods are also useful in protecting plants against any Diabroticaplant pest including, for example, Diabrotica adelpha; Diabroticaamecameca; Diabrotica balteata; Diabrotica barberi; Diabroticabiannularis; Diabrotica cristata; Diabrotica decempunctata; Diabroticadissimilis; Diabrotica lemniscata; Diabrotica limitata (including, forexample, Diabrotica limitata quindecimpuncata); Diabrotica longicornis;Diabrotica nummularis; Diabrotica porracea; Diabrotica scutellata;Diabrotica sexmaculata; Diabrotica speciosa (including, for example,Diabrotica speciosa speciosa); Diabrotica tibialis; Diabroticaundecimpunctata (including, for example, Southern corn rootworm(Diabrotica undecimpunctata), Diabrotica undecimpunctata duodecimnotata;Diabrotica undecimpunctata howardi (spotted cucumber beetle); Diabroticaundecimpunctata undecimpunctata (western spotted cucumber beetle));Diabrotica virgifera (including, for example, Diabrotica virgiferavirgifera (western corn rootworm) and Diabrotica virgifera zeae (Mexicancorn rootworm)); Diabrotica viridula; Diabrotica wartensis; Diabroticasp. JJG335; Diabrotica sp. JJG336; Diabrotica sp. JJG341; Diabrotica sp.JJG356; Diabrotica sp. JJG362; and, Diabrotica sp. JJG365.

In certain embodiments, the Diabrotica plant pest comprises D. virgiferavirgifera, D. barberi, D. virgifera zeae, D. speciosa, or D.undecimpunctata howardi.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guende (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenće (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermuller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guërin-Mëneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Mëneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guende; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Gëhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifolii Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus St∪l (rice leafhopper); Nilaparvatalugens Stal (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schaffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Insect pests of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

II. Target Sequences

As used herein, a “target sequence” or “target polynucleotide” comprisesany sequence in the pest that one desires to reduce the level ofexpression thereof. In certain embodiments, decreasing the level ofexpression of the target sequence in the pest controls the pest. Forinstance, the target sequence may be essential for growth anddevelopment. Non-limiting examples of target sequences include apolynucleotide set forth in SEQ ID NOS.: 1-53 or 107-254, or variantsand fragments thereof, and complements thereof. As exemplified elsewhereherein, decreasing the level of expression of one or more of thesetarget sequences in a Coleopteran plant pest or a Diabrotica plant pestcontrols the pest.

III. Silencing Elements

By “silencing element” is intended a polynucleotide which when contactedby or ingested by a plant insect pest, is capable of reducing oreliminating the level or expression of a target polynucleotide or thepolypeptide encoded thereby. Accordingly, it is to be understood that“silencing element,” as used herein, comprises polynucleotides such asRNA constructs, double stranded RNA (dsRNA), hairpin RNA, and senseand/or antisense RNA. In one embodiment, the silencing element employedcan reduce or eliminate the expression level of the target sequence byinfluencing the level of the target RNA transcript or, alternatively, byinfluencing translation and thereby affecting the level of the encodedpolypeptide. Methods to assay for functional silencing elements that arecapable of reducing or eliminating the level of a sequence of interestare disclosed elsewhere herein. A single polynucleotide employed in thedisclosed methods can comprise one or more silencing elements to thesame or different target polynucleotides. The silencing element can beproduced in vivo (i.e., in a host cell such as a plant or microorganism)or in vitro.

In certain embodiments, a silencing element may comprise a chimericconstruction molecule comprising two or more disclosed sequences orportions thereof. For example, the chimeric construction may be ahairpin or dsRNA as disclosed herein. A chimera may comprise two or moredisclosed sequences or portions thereof. In one embodiment, a chimeracontemplates two complementary sequences set forth herein, or portionsthereof, having some degree of mismatch between the complementarysequences such that the two sequences are not perfect complements of oneanother. Providing at least two different sequences in a singlesilencing element may allow for targeting multiple genes using onesilencing element and/or for example, one expression cassette. Targetingmultiple genes may allow for slowing or reducing the possibility ofresistance by the pest. In addition, providing multiple targetingability in one expressed molecule may reduce the expression burden ofthe transformed plant or plant product, or provide topical treatmentsthat are capable of targeting multiple hosts with one application.

In certain embodiments, while the silencing element controls pests,preferably the silencing element has no effect on the normal plant orplant part.

As discussed in further detail below, silencing elements can include,but are not limited to, a sense suppression element, an antisensesuppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA,or a hairpin suppression element. In an embodiment, silencing elementsmay comprise a chimera where two or more disclosed sequences or activefragments or variants, or complements thereof, are found in the same RNAmolecule. In various embodiments, a disclosed sequence or activefragment or variant, or complement thereof, may be present as more thanone copy in a DNA construct, silencing element, DNA molecule or RNAmolecule. In a hairpin or dsRNA molecule, the location of a sense orantisense sequence in the molecule, for example, in which sequence istranscribed first or is located on a particular terminus of the RNAmolecule, is not limiting to the disclosed sequences, and the dsRNA isnot to be limited by disclosures herein of a particular location forsuch a sequence. Non-limiting examples of silencing elements that can beemployed to decrease expression of these target sequences comprisefragments or variants of the sense or antisense sequence, oralternatively consists of the sense or antisense sequence, of a sequenceset forth in SEQ ID NOS.: 1-53 or 107-254, or variants and fragmentsthereof, and complements thereof. The silencing element can furthercomprise additional sequences that advantageously effect transcriptionand/or the stability of a resulting transcript. For example, thesilencing elements can comprise at least one thymine residue at the 3′end. This can aid in stabilization. Thus, the silencing elements canhave at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more thymine residues atthe 3′ end. As discussed in further detail below, enhancer suppressorelements can also be employed in conjunction with the silencing elementsdisclosed herein.

By “reduces” or “reducing” the expression level of a polynucleotide or apolypeptide encoded thereby is intended to mean, the polynucleotide orpolypeptide level of the target sequence is statistically lower than thepolynucleotide level or polypeptide level of the same target sequence inan appropriate control pest which is not exposed to (i.e., has notingested or come into contact with) the silencing element. In particularembodiments, methods and/or compositions disclosed herein reduce thepolynucleotide level and/or the polypeptide level of the target sequencein a plant insect pest to less than 95%, less than 90%, less than 80%,less than 70%, less than 60%, less than 50%, less than 40%, less than30%, less than 20%, less than 10%, or less than 5% of the polynucleotidelevel, or the level of the polypeptide encoded thereby, of the sametarget sequence in an appropriate control pest.

In some embodiments, a silencing element has substantial sequenceidentity to the target polynucleotide, typically greater than about 65%sequence identity, greater than about 85% sequence identity, about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.Furthermore, a silencing element can be complementary to a portion ofthe target polynucleotide. Generally, sequences of at least 15, 16, 17,18, 19, 20, 22, 25, 50, 100, 200, 300, 400, 450 continuous nucleotidesor greater of the sequence set forth in any of SEQ ID NOS.: 1-53 or107-254, or variants and fragments thereof, and complements thereof maybe used. Methods to assay for the level of the RNA transcript, the levelof the encoded polypeptide, or the activity of the polynucleotide orpolypeptide are discussed elsewhere herein.

i. Sense Suppression Elements

As used herein, a “sense suppression element” comprises a polynucleotidedesigned to express an RNA molecule corresponding to at least a part ofa target messenger RNA in the “sense” orientation. Expression of the RNAmolecule comprising the sense suppression element reduces or eliminatesthe level of the target polynucleotide or the polypeptide encodedthereby. The polynucleotide comprising the sense suppression element maycorrespond to all or part of the sequence of the target polynucleotide,all or part of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the coding sequence of the targetpolynucleotide, or all or part of both the coding sequence and theuntranslated regions of the target polynucleotide.

Typically, a sense suppression element has substantial sequence identityto the target polynucleotide, typically greater than about 65% sequenceidentity, greater than about 85% sequence identity, about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. See, U.S. Pat.Nos. 5,283,184 and 5,034,323; herein incorporated by reference. Thesense suppression element can be any length so long as it allows for thesuppression of the targeted sequence. The sense suppression element canbe, for example, 15, 16, 17, 18, 19, 20, 22, 25, 30, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300nucleotides or longer of the target polynucleotides set forth in any ofSEQ ID NOS.: 1-53 or 107-254, or variants and fragments thereof, andcomplements thereof. In other embodiments, the sense suppression elementcan be, for example, about 15-25, 19-35, 19-50, 25-100, 100-150,150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the targetpolynucleotides set forth in any of SEQ ID NOS.: 1-53 or 107-254, orvariants and fragments thereof, and complements thereof.

ii. Antisense Suppression Elements

As used herein, an “antisense suppression element” comprises apolynucleotide which is designed to express an RNA moleculecomplementary to all or part of a target messenger RNA. Expression ofthe antisense RNA suppression element reduces or eliminates the level ofthe target polynucleotide. The polynucleotide for use in antisensesuppression may correspond to all or part of the complement of thesequence encoding the target polynucleotide, all or part of thecomplement of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the complement of the coding sequence ofthe target polynucleotide, or all or part of the complement of both thecoding sequence and the untranslated regions of the targetpolynucleotide. In addition, the antisense suppression element may befully complementary (i.e., 100% identical to the complement of thetarget sequence) or partially complementary (i.e., less than 100%identical to the complement of the target sequence) to the targetpolynucleotide. In certain embodiments, the antisense suppressionelement comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence complementarity to the target polynucleotide.Antisense suppression may be used to inhibit the expression of multipleproteins in the same plant. See, for example, U.S. Pat. No. 5,942,657.Furthermore, the antisense suppression element can be complementary to aportion of the target polynucleotide. Generally, sequences of at least15, 16, 17, 18, 19, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotidesor greater of the sequence set forth in any of SEQ ID NOS.: 1-53 or107-254, or variants and fragments thereof, and complements thereof maybe used. Methods for using antisense suppression to inhibit theexpression of endogenous genes in plants are described, for example, inLiu et al (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. No.5,942,657, which is herein incorporated by reference.

iii. Double Stranded RNA Suppression Element

A “double stranded RNA silencing element” or “dsRNA,” comprises at leastone transcript that is capable of forming a dsRNA either before or afteringestion by a plant insect pest. Thus, a “dsRNA silencing element”includes a dsRNA, a transcript or polyribonucleotide capable of forminga dsRNA or more than one transcript or polyribonucleotide capable offorming a dsRNA. “Double stranded RNA” or “dsRNA” refers to apolyribonucleotide structure formed either by a singleself-complementary RNA molecule or a polyribonucleotide structure formedby the expression of at least two distinct RNA strands. The dsRNAmolecule(s) employed in the disclosed methods and compositions mediatethe reduction of expression of a target sequence, for example, bymediating RNA interference “RNAi” or gene silencing in asequence-specific manner. In various embodiments, the dsRNA is capableof reducing or eliminating the level or expression of a targetpolynucleotide or the polypeptide encoded thereby in a plant insectpest.

The dsRNA can reduce or eliminate the expression level of the targetsequence by influencing the level of the target RNA transcript, byinfluencing translation and thereby affecting the level of the encodedpolypeptide, or by influencing expression at the pre-transcriptionallevel (i.e., via the modulation of chromatin structure, methylationpattern, etc., to alter gene expression). For example, see Verdel et al.(2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science303:669-672; Allshire (2002) Science 297:1818-1819; Volpe et al. (2002)Science 297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hallet al. (2002) Science 297:2232-2237. Methods to assay for functionaldsRNA that are capable of reducing or eliminating the level of asequence of interest are disclosed elsewhere herein. Accordingly, asused herein, the term “dsRNA” is meant to encompass other terms used todescribe nucleic acid molecules that are capable of mediating RNAinterference or gene silencing, including, for example,short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptionalgene silencing RNA (ptgsRNA), and others.

In certain embodiments, at least one strand of the duplex ordouble-stranded region of the dsRNA shares sufficient sequence identityor sequence complementarity to the target polynucleotide to allow thedsRNA to reduce the level of expression of the target sequence. In someembodiments, a dsRNA has substantial sequence identity to the targetpolynucleotide, typically greater than about 65% sequence identity,greater than about 85% sequence identity, about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity. Furthermore, a dsRNAelement can be complementary to a portion of the target polynucleotide.Generally, sequences of at least 15, 16, 17, 18, 19, 20, 22, 25, 50,100, 200, 300, 400, 450 nucleotides or greater of the sequence set forthin any of SEQ ID NOS.: 1-53 or 107-254, or variants and fragmentsthereof, and complements thereof may be used. As used herein, the strandthat is complementary to the target polynucleotide is the “antisensestrand” and the strand homologous to the target polynucleotide is the“sense strand.”

In another embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNAcomprises an RNA molecule that is capable of folding back onto itself toform a double stranded structure. Multiple structures can be employed ashairpin elements. In certain embodiments, the dsRNA suppression elementcomprises a hairpin element which comprises in the following order, afirst segment, a second segment, and a third segment, where the firstand the third segment share sufficient complementarity to allow thetranscribed RNA to form a double-stranded stem-loop structure.

The “second segment” of the hairpin comprises a “loop” or a “loopregion.” These terms are used synonymously herein and are to beconstrued broadly to comprise any nucleotide sequence that confersenough flexibility to allow self-pairing to occur between complementaryregions of a polynucleotide (i.e., segments 1 and 3 which form the stemof the hairpin). For example, in some embodiments, the loop region maybe substantially single stranded and act as a spacer between theself-complementary regions of the hairpin stem-loop. In someembodiments, the loop region can comprise a random or nonsensenucleotide sequence and thus not share sequence identity to a targetpolynucleotide. In other embodiments, the loop region comprises a senseor an antisense RNA sequence or fragment thereof that shares identity toa target polynucleotide. See, for example, International PatentPublication No. WO 02/00904. In certain embodiments, the loop sequencecan include an intron sequence, a sequence derived from an intronsequence, a sequence homologous to an intron sequence, or a modifiedintron sequence. The intron sequence can be one found in the same or adifferent species from which segments 1 and 3 are derived. In certainembodiments, the loop region can be optimized to be as short as possiblewhile still providing enough intramolecular flexibility to allow theformation of the base-paired stem region. Accordingly, the loop sequenceis generally less than 1000, 900, 800, 700, 600, 500, 400, 300, 200,100, 50, 25, 20, 19, 18, 17, 16, 15, 10 nucleotides or less.

The “first” and the “third” segment of the hairpin RNA molecule comprisethe base-paired stem of the hairpin structure. The first and the thirdsegments are inverted repeats of one another and share sufficientcomplementarity to allow the formation of the base-paired stem region.In certain embodiments, the first and the third segments are fullycomplementary to one another. Alternatively, the first and the thirdsegment may be partially complementary to each other so long as they arecapable of hybridizing to one another to form a base-paired stem region.The amount of complementarity between the first and the third segmentcan be calculated as a percentage of the entire segment. Thus, the firstand the third segment of the hairpin RNA generally share at least 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, upto and including 100% complementarity.

The first and the third segment are at least about 1000, 500, 475, 450,425, 400, 375, 350, 325, 300, 250, 225, 200, 175, 150, 125, 100, 75, 60,50, 40, 30, 25, 22, 20, 19, 18, 17, 16, 15 or 10 nucleotides in length.In certain embodiments, the length of the first and/or the third segmentis about 10-100 nucleotides, about 10 to about 75 nucleotides, about 10to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 toabout 35 nucleotides, about 10 to about 30 nucleotides, about 10 toabout 25 nucleotides, about 10 to about 19 nucleotides, about 10 toabout 20 nucleotides, about 19 to about 50 nucleotides, about 50nucleotides to about 100 nucleotides, about 100 nucleotides to about 150nucleotides, about 100 nucleotides to about 300 nucleotides, about 150nucleotides to about 200 nucleotides, about 200 nucleotides to about 250nucleotides, about 250 nucleotides to about 300 nucleotides, about 300nucleotides to about 350 nucleotides, about 350 nucleotides to about 400nucleotides, about 400 nucleotide to about 500 nucleotides, about 600nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, about 1100nt, about 1200 nt, 1300 nt, 1400 nt, 1500 nt, 1600 nt, 1700 nt, 1800 nt,1900 nt, 2000 nt or longer. In other embodiments, the length of thefirst and/or the third segment comprises at least 10-19 nucleotides,10-20 nucleotides; 19-35 nucleotides, 20-35 nucleotides; 30-45nucleotides; 40-50 nucleotides; 50-100 nucleotides; 100-300 nucleotides;about 500-700 nucleotides; about 700-900 nucleotides; about 900-1100nucleotides; about 1300-1500 nucleotides; about 1500-1700 nucleotides;about 1700-1900 nucleotides; about 1900-2100 nucleotides; about2100-2300 nucleotides; or about 2300-2500 nucleotides. See, for example,International Publication No. WO 02/00904.

The disclosed hairpin molecules or double-stranded RNA molecules mayhave more than one disclosed sequence or active fragments or variants,or complements thereof, found in the same portion of the RNA molecule.For example, in a chimeric hairpin structure, the first segment of ahairpin molecule comprises two polynucleotide sections, each with adifferent disclosed sequence. For example, reading from one terminus ofthe hairpin, the first segment is composed of sequences from twoseparate genes (A followed by B). This first segment is followed by thesecond segment, the loop portion of the hairpin. The loop segment isfollowed by the third segment, where the complementary strands of thesequences in the first segment are found (B* followed by A*) in formingthe stem-loop, hairpin structure, the stem contains SeqA-A* at thedistal end of the stem and SeqB-B* proximal to the loop region.

In certain embodiments, the first and the third segment comprise atleast 20 nucleotides having at least 85% complementary to the firstsegment. In still other embodiments, the first and the third segmentswhich form the stem-loop structure of the hairpin comprise 3′ or 5′overhang regions having unpaired nucleotide residues.

In certain embodiments, the sequences used in the first, the second,and/or the third segments comprise domains that are designed to havesufficient sequence identity to a target polynucleotide of interest andthereby have the ability to decrease the level of expression of thetarget polynucleotide. The specificity of the inhibitory RNA transcriptsis therefore generally conferred by these domains of the silencingelement. Thus, in some embodiments, the first, second and/or thirdsegment of the silencing element comprise a domain having at least 10,at least 15, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 30, at least 40, at least50, at least 100, at least 200, at least 300, at least 500, at least1000, or more than 1000 nucleotides that share sufficient sequenceidentity to the target polynucleotide to allow for a decrease inexpression levels of the target polynucleotide when expressed in anappropriate cell. In other embodiments, the domain is between about 15to 50 nucleotides, about 19-35 nucleotides, about 20-35 nucleotides,about 25-50 nucleotides, about 19 to 75 nucleotides, about 20 to 75nucleotides, about 40-90 nucleotides about 15-100 nucleotides, 10-100nucleotides, about 10 to about 75 nucleotides, about 10 to about 50nucleotides, about 10 to about 40 nucleotides, about 10 to about 35nucleotides, about 10 to about 30 nucleotides, about 10 to about 25nucleotides, about 10 to about 20 nucleotides, about 10 to about 19nucleotides, about 50 nucleotides to about 100 nucleotides, about 100nucleotides to about 150 nucleotides, about 150 nucleotides to about 200nucleotides, about 200 nucleotides to about 250 nucleotides, about 250nucleotides to about 300 nucleotides, about 300 nucleotides to about 350nucleotides, about 350 nucleotides to about 400 nucleotides, about 400nucleotide to about 500 nucleotides or longer. In other embodiments, thelength of the first and/or the third segment comprises at least 10-20nucleotides, at least 10-19 nucleotides, 20-35 nucleotides, 30-45nucleotides, 40-50 nucleotides, 50-100 nucleotides, or about 100-300nucleotides.

In certain embodiments, a domain of the first, the second, and/or thethird segment has 100% sequence identity to the target polynucleotide.In other embodiments, the domain of the first, the second and/or thethird segment having homology to the target polynucleotide have at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or greater sequence identity to a region of the targetpolynucleotide. The sequence identity of the domains of the first, thesecond and/or the third segments complementary to a targetpolynucleotide need only be sufficient to decrease expression of thetarget polynucleotide of interest. See, for example, Chuang andMeyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijket al. (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell(2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMC Biotechnology3:7, and U.S. Patent Publication No. 20030175965; each of which isherein incorporated by reference. A transient assay for the efficiencyof hpRNA constructs to silence gene expression in vivo has beendescribed by Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140.

The amount of complementarity shared between the first, second, and/orthird segment and the target polynucleotide or the amount ofcomplementarity shared between the first segment and the third segment(i.e., the stem of the hairpin structure) may vary depending on theorganism in which gene expression is to be controlled. Some organisms orcell types may require exact pairing or 100% identity, while otherorganisms or cell types may tolerate some mismatching. In some cells,for example, a single nucleotide mismatch in the targeting sequenceabrogates the ability to suppress gene expression. In these cells, thedisclosed suppression cassettes can be used to target the suppression ofmutant genes, for example, oncogenes whose transcripts comprise pointmutations and therefore they can be specifically targeted using themethods and compositions disclosed herein without altering theexpression of the remaining wild-type allele. In other organisms,holistic sequence variability may be tolerated as long as some 22 ntregion of the sequence is represented in 100% homology between targetpolynucleotide and the suppression cassette.

Any region of the target polynucleotide can be used to design a domainof the silencing element that shares sufficient sequence identity toallow expression of the hairpin transcript to decrease the level of thetarget polynucleotide. For instance, a domain may be designed to sharesequence identity to the 5′ untranslated region of the targetpolynucleotide(s), the 3′ untranslated region of the targetpolynucleotide(s), exonic regions of the target polynucleotide(s),intronic regions of the target polynucleotide(s), and any combinationthereof. In certain embodiments, a domain of the silencing elementshares sufficient identity, homology, or is complementary to at leastabout 15, 16, 17, 18, 19, 20, 22, 25 or 30 consecutive nucleotides fromabout nucleotides 1-50, 25-75, 75-125, 50-100, 125-175, 175-225,100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425,300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625,550-600, 625-675, 675-725, 600-650, 625-675, 675-725, 650-700, 725-825,825-875, 750-800, 875-925, 925-975, 850-900, 925-975, 975-1025,950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175,1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425,1300-1400, 1425-1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625,1625-1675, 1675-1725, 1725-1775, 1775-1825, 1825-1875, 1875-1925,1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175-2225,1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of the targetsequence. In some instances to optimize the siRNA sequences employed inthe hairpin, the synthetic oligodeoxyribonucleotide/RNAse H method canbe used to determine sites on the target mRNA that are in a conformationthat is susceptible to RNA silencing. See, for example, Vickers et al.(2003) J. Biol. Chem 278:7108-7118 and Yang et al. (2002) Proc. Natl.Acad. Sci. USA 99:9442-9447, herein incorporated by reference. Thesestudies indicate that there is a significant correlation between theRNase-H-sensitive sites and sites that promote efficient siRNA-directedmRNA degradation.

The hairpin silencing element may also be designed such that the sensesequence or the antisense sequence do not correspond to a targetpolynucleotide. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the target polynucleotide. Thus, it is the loop regionthat determines the specificity of the RNA interference. See, forexample, WO 02/00904.

In addition, transcriptional gene silencing (TGS) may be accomplishedthrough use of a hairpin suppression element where the inverted repeatof the hairpin shares sequence identity with the promoter region of atarget polynucleotide to be silenced. See, for example, Aufsatz et al.(2002) PNAS 99 (Suppl. 4):16499-16506 and Mette et al. (2000) EMBO J19(19):5194-5201.

In other embodiments, the silencing element can comprise a small RNA(sRNA). sRNAs can comprise both micro RNA (miRNA) and short-interferingRNA (siRNA) (Meister and Tuschl (2004) Nature 431:343-349 and Bonetta etal. (2004) Nature Methods 1:79-86). miRNAs are regulatory agentscomprising about 19 to about 24 ribonucleotides in length which arehighly efficient at inhibiting the expression of target polynucleotides.See, for example Javier et al. (2003) Nature 425: 257-263. For miRNAinterference, the silencing element can be designed to express a dsRNAmolecule that forms a hairpin structure or partially base-pairedstructure containing a 19, 20, 21, 22, 23, 24 or 25 nucleotide sequencethat is complementary to the target polynucleotide of interest. ThemiRNA can be synthetically made, or transcribed as a longer RNA which issubsequently cleaved to produce the active miRNA. Specifically, themiRNA can comprise 19 nucleotides of the sequence having homology to atarget polynucleotide in sense orientation and 19 nucleotides of acorresponding antisense sequence that is complementary to the sensesequence. The miRNA can be an “artificial miRNA” or “amiRNA” whichcomprises a miRNA sequence that is synthetically designed to silence atarget sequence.

When expressing an miRNA the final (mature) miRNA is present in a duplexin a precursor backbone structure, the two strands being referred to asthe miRNA (the strand that will eventually base pair with the target)and miRNA*(star sequence). It has been demonstrated that miRNAs can betransgenically expressed and target genes of interest for efficientsilencing (Highly specific gene silencing by artificial microRNAs inArabidopsis Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D.Plant Cell. 2006 May; 18(5):1121-33. Epub 2006 Mar. 10; and Expressionof artificial microRNAs in transgenic Arabidopsis thaliana confers virusresistance. Niu Q W, Lin S S, Reyes J L, Chen K C, Wu H W, Yeh S D, ChuaN H. Nat Biotechnol. 2006 November; 24(11):1420-8. Epub 2006 Oct. 22.Erratum in: Nat Biotechnol. 2007 February; 25(2):254.).

The silencing element for miRNA interference comprises a miRNA primarysequence. The miRNA primary sequence comprises a DNA sequence having themiRNA and star sequences separated by a loop as well as additionalsequences flanking this region that are important for processing. Whenexpressed as an RNA, the structure of the primary miRNA is such as toallow for the formation of a hairpin RNA structure that can be processedinto a mature miRNA. In some embodiments, the miRNA backbone comprises agenomic or cDNA miRNA precursor sequence, wherein said sequencecomprises a native primary in which a heterologous (artificial) maturemiRNA and star sequence are inserted.

As used herein, a “star sequence” is the sequence within a miRNAprecursor backbone that is complementary to the miRNA and forms a duplexwith the miRNA to form the stem structure of a hairpin RNA. In someembodiments, the star sequence can comprise less than 100%complementarity to the miRNA sequence. Alternatively, the star sequencecan comprise at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or lowersequence complementarity to the miRNA sequence as long as the starsequence has sufficient complementarity to the miRNA sequence to form adouble stranded structure. In still further embodiments, the starsequence comprises a sequence having 1, 2, 3, 4, 5 or more mismatcheswith the miRNA sequence and still has sufficient complementarity to forma double stranded structure with the miRNA sequence resulting inproduction of miRNA and suppression of the target sequence.

The miRNA precursor backbones can be from any plant. In someembodiments, the miRNA precursor backbone is from a monocot. In otherembodiments, the miRNA precursor backbone is from a dicot. In furtherembodiments, the backbone is from maize or soybean. MicroRNA precursorbackbones have been described previously. For example, US20090155910A1(WO 2009/079532) discloses the following soybean miRNA precursorbackbones: 156c, 159, 166b, 168c, 396b and 398b, and US20090155909A1 (WO2009/079548) discloses the following maize miRNA precursor backbones:159c, 164h, 168a, 169r, and 396h.

Thus, the primary miRNA can be altered to allow for efficient insertionof heterologous miRNA and star sequences within the miRNA precursorbackbone. In such instances, the miRNA segment and the star segment ofthe miRNA precursor backbone are replaced with the heterologous miRNAand the heterologous star sequences, designed to target any sequence ofinterest, using a PCR technique and cloned into an expression construct.It is recognized that there could be alterations to the position atwhich the artificial miRNA and star sequences are inserted into thebackbone. Detailed methods for inserting the miRNA and star sequenceinto the miRNA precursor backbone are described in, for example, USPatent Applications 20090155909A1 and US20090155910A1.

When designing a miRNA sequence and star sequence, various designchoices can be made. See, for example, Schwab R, et al. (2005) Dev Cell8: 517-27. In non-limiting embodiments, the miRNA sequences disclosedherein can have a “U” at the 5′-end, a “C” or “G” at the 19th nucleotideposition, and an “A” or “U” at the 10th nucleotide position. In otherembodiments, the miRNA design is such that the miRNA have a high freedelta-G as calculated using the ZipFold algorithm (Markham, N. R. &Zuker, M. (2005) Nucleic Acids Res. 33: W577-W581.) Optionally, a onebase pair change can be added within the 5′ portion of the miRNA so thatthe sequence differs from the target sequence by one nucleotide.

The methods and compositions disclosed herein employ DNA constructs thatwhen transcribed “form” a silencing element, such as a dsRNA molecule.The methods and compositions also may comprise a host cell comprisingthe DNA construct encoding a silencing element. In another embodiment,The methods and compositions also may comprise a transgenic plantcomprising the DNA construct encoding a silencing element. Accordingly,the heterologous polynucleotide being expressed need not form the dsRNAby itself, but can interact with other sequences in the plant cell or inthe pest gut after ingestion to allow the formation of the dsRNA. Forexample, a chimeric polynucleotide that can selectively silence thetarget polynucleotide can be generated by expressing a chimericconstruct comprising the target sequence for a miRNA or siRNA to asequence corresponding to all or part of the gene or genes to besilenced. In this embodiment, the dsRNA is “formed” when the target forthe miRNA or siRNA interacts with the miRNA present in the cell. Theresulting dsRNA can then reduce the level of expression of the gene orgenes to be silenced. See, for example, US Application Publication2007-0130653, entitled “Methods and Compositions for Gene Silencing”.The construct can be designed to have a target for an endogenous miRNAor alternatively, a target for a heterologous and/or synthetic miRNA canbe employed in the construct. If a heterologous and/or synthetic miRNAis employed, it can be introduced into the cell on the same nucleotideconstruct as the chimeric polynucleotide or on a separate construct. Asdiscussed elsewhere herein, any method can be used to introduce theconstruct comprising the heterologous miRNA.

IV. Variants and Fragments

By “fragment” is intended a portion of the polynucleotide or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a polynucleotide may encode protein fragments that retain thebiological activity of the native protein. Alternatively, fragments of apolynucleotide that are useful as a silencing element do not need toencode fragment proteins that retain biological activity. Thus,fragments of a nucleotide sequence may range from at least about 10,about 15, about 16, about 17, about 18, about 19, nucleotides, about 20nucleotides, about 22 nucleotides, about 50 nucleotides, about 75nucleotides, about 100 nucleotides, 200 nucleotides, 300 nucleotides,400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides andup to and including one nucleotide less than the full-lengthpolynucleotide employed. Alternatively, fragments of a nucleotidesequence may range from 1-50, 25-75, 75-125, 50-100, 125-175, 175-225,100-150, 100-300, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375,375-425, 300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575,575-625, 550-600, 625-675, 675-725, 600-650, 625-675, 675-725, 650-700,725-825, 825-875, 750-800, 875-925, 925-975, 850-900, 925-975, 975-1025,950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175,1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425,1300-1400, 1425-1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625,1625-1675, 1675-1725, 1725-1775, 1775-1825, 1825-1875, 1875-1925,1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175-2225,1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of any one of SEQID NOS.: 1-53 or 107-254, or variants and fragments thereof, andcomplements thereof. Methods to assay for the activity of a desiredsilencing element are described elsewhere herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. A variant of apolynucleotide that is useful as a silencing element will retain theability to reduce expression of the target polynucleotide and, in someembodiments, thereby control a plant insect pest of interest. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the disclosed polypeptides. Variant polynucleotidesalso include synthetically derived polynucleotide, such as thosegenerated, for example, by using site-directed mutagenesis, but continueto retain the desired activity. Generally, variants of a particulardisclosed polynucleotide (i.e., a silencing element) will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular disclosed polynucleotide (i.e., the referencepolynucleotide) can also be evaluated by comparison of the percentsequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of disclosedpolynucleotides employed is evaluated by comparison of the percentsequence identity shared by the two polypeptides they encode, thepercent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

A method is further provided for identifying a silencing element fromthe target polynucleotides set forth in SEQ ID NOS.: 1-53 or 107-254, orvariants and fragments thereof, and complements thereof. Such methodscomprise obtaining a candidate fragment of any one of SEQ ID NOS.: 1-53or 107-254, or variants and fragments thereof, and complements thereof,which is of sufficient length to act as a silencing element and therebyreduce the expression of the target polynucleotide and/or control adesired pest; expressing said candidate polynucleotide fragment in anappropriate expression cassette to produce a candidate silencing elementand determining is said candidate polynucleotide fragment has theactivity of a silencing element and thereby reduce the expression of thetarget polynucleotide and/or controls a desired pest. Methods ofidentifying such candidate fragments based on the desired pathway forsuppression, in light of the teachings provided herein, are known. Forexample, various bioinformatics programs can be employed to identify theregion of the target polynucleotides that could be exploited to generatea silencing element. See, for example, Elbahir et al. (2001) Genes andDevelopment 15:188-200, Schwartz et al. (2003) Cell 115:199-208,Khvorova et al. (2003) Cell 115:209-216. See also, siRNA at Whitehead(jura.wi.mit.edu/bioc/siRNAext/) which calculates the binding energiesfor both sense and antisense siRNAs. See, alsogenscript.com/ssl-bin/app/rnai?op=known; Block-iT™ RNAi designer fromInvitrogen and GenScript siRNA Construct Builder. In various aspects, itis to be understand that the term “ . . . SEQ ID NOS.: 1-53 or 107-254,or variants or fragments thereof, or complements thereof . . . ” isintended to mean that the disclosed sequences comprise SEQ ID NOS.: 1-53or 107-254, and/or fragments of SEQ ID NOS.: 1-53 or 107-254, and/orvariants of SEQ ID NOS.: 1-53 or 107-254, and/or the complements of SEQID NOS.: 1-53 or 107-254, the variants of SEQ ID NOS.: 1-53 or 107-254,and/or the fragments of SEQ ID NOS.: 1-53 or 107-254, individually (or)or inclusive of some or all listed sequences.

V. DNA Constructs

The use of the term “polynucleotide” is not intended to be limiting topolynucleotides comprising DNA. Those of ordinary skill in the art willrecognize that polynucleotides can comprise ribonucleotides andcombinations of ribonucleotides and deoxyribonucleotides. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The disclosedpolynucleotides also encompass all forms of sequences including, but notlimited to, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures, and the like.

The polynucleotide encoding the silencing element or in certainembodiments employed in the disclosed methods and compositions can beprovided in expression cassettes for expression in a plant or organismof interest. It is recognized that multiple silencing elements includingmultiple identical silencing elements, multiple silencing elementstargeting different regions of the target sequence, or multiplesilencing elements from different target sequences can be used. In thisembodiment, it is recognized that each silencing element may be encodedby a single or separate cassette, DNA construct, or vector. Asdiscussed, any means of providing the silencing element is contemplated.A plant or plant cell can be transformed with a single cassettecomprising DNA encoding one or more silencing elements or separatecassettes encoding a silencing element can be used to transform a plantor plant cell or host cell. Likewise, a plant transformed with onecomponent can be subsequently transformed with the second component. Oneor more DNA constructs encoding silencing elements can also be broughttogether by sexual crossing. That is, a first plant comprising onecomponent is crossed with a second plant comprising the secondcomponent. Progeny plants from the cross will comprise both components.

The expression cassette can include 5′ and 3′ regulatory sequencesoperably linked to the polynucleotide of the invention. “Operablylinked” is intended to mean a functional linkage between two or moreelements. For example, an operable linkage between a polynucleotide ofthe invention and a regulatory sequence (i.e., a promoter) is afunctional link that allows for expression of the polynucleotidedisclosed herein. Operably linked elements may be contiguous ornon-contiguous. When used to refer to the joining of two protein codingregions, by operably linked is intended that the coding regions are inthe same reading frame. The cassette may additionally contain at leastone additional polynucleotide to be cotransformed into the organism.Alternatively, the additional polypeptide(s) can be provided on multipleexpression cassettes. Expression cassettes can be provided with aplurality of restriction sites and/or recombination sites for insertionof the polynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide encoding the silencing elementemployed in the methods and compositions of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants. In other embodiment, the double strandedRNA is expressed from a suppression cassette. Such a cassette cancomprise two convergent promoters that drive transcription of anoperably linked silencing element. “Convergent promoters” refers topromoters that are oriented on either terminus of the operably linkedpolynucleotide encoding the silencing element such that each promoterdrives transcription of the silencing element in opposite directions,yielding two transcripts. In such embodiments, the convergent promotersallow for the transcription of the sense and anti-sense strand and thusallow for the formation of a dsRNA. Such a cassette may also comprisetwo divergent promoters that drive transcription of one or more operablylinked polynucleotides encoding the silencing elements. “Divergentpromoters” refers to promoters that are oriented in opposite directionsof each other, driving transcription of the one or more polynucleotidesencoding the silencing elements in opposite directions. In suchembodiments, the divergent promoters allow for the transcription of thesense and antisense strands and allow for the formation of a dsRNA. Insuch embodiments, the divergent promoters also allow for thetranscription of at least two separate hairpin RNAs. In anotherembodiment, one cassette comprising two or more polynucleotides encodingthe silencing elements under the control of two separate promoters inthe same orientation is present in a construct. In another embodiment,two or more individual cassettes, each comprising at least onepolynucleotide encoding the silencing element under the control of apromoter, are present in a construct in the same orientation.

The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or thepolynucleotides disclosed herein may be native/analogous to the hostcell or to each other. Alternatively, the regulatory regions and/or thepolynucleotide disclosed herein may be heterologous to the host cell orto each other. As used herein, “heterologous” in reference to a sequenceis a sequence that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. As used herein, a chimeric gene comprises a codingsequence operably linked to a transcription initiation region that isheterologous to the coding sequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked polynucleotide encodingthe silencing element, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the polynucleotide encoding the silencing element, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible, orother promoters for expression in the host organism.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. An inducible promoter, for instance, apathogen-inducible promoter could also be employed. Such promotersinclude those from pathogenesis-related proteins (PR proteins), whichare induced following infection by a pathogen; e.g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfiet al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116.See also WO 99/43819.

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and VanLoon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible). Of particular interest is the inducible promoterfor the maize PRms gene, whose expression is induced by the pathogenFusarium moniliforme (see, for example, Cordero et al. (1992) Physiol.Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-la promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters includeYamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); and milps (myo-inositol-1-phosphate synthase) (see U.S. Pat. No.6,225,529, herein incorporated by reference). Gamma-zein and Glob-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, bean D-phaseolin, napin, □-conglycinin,soybean lectin, cruciferin, and the like. For monocots, seed-specificpromoters include, but are not limited to, maize 15 kDa zein, 22 kDazein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,etc. See also WO 00/12733, where seed-preferred promoters from end1 andend2 genes are disclosed. A promoter that has “preferred” expression ina particular tissue is expressed in that tissue to a greater degree thanin at least one other plant tissue. Some tissue-preferred promoters showexpression almost exclusively in the particular tissue.

In an embodiment, the plant-expressed promoter is a vascular-specificpromoter such as a phloem-specific promoter. A “vascular-specific”promoter, as used herein, is a promoter which is at least expressed invascular cells, or a promoter which is preferentially expressed invascular cells. Expression of a vascular-specific promoter need not beexclusively in vascular cells, expression in other cell types or tissuesis possible. A “phloem-specific promoter” as used herein, is aplant-expressible promoter which is at least expressed in phloem cells,or a promoter which is preferentially expressed in phloem cells.

Expression of a phloem-specific promoter need not be exclusively inphloem cells, expression in other cell types or tissues, e.g., xylemtissue, is possible. In one embodiment of this invention, aphloem-specific promoter is a plant-expressible promoter at leastexpressed in phloem cells, wherein the expression in non-phloem cells ismore limited (or absent) compared to the expression in phloem cells.Examples of suitable vascular-specific or phloem-specific promoters inaccordance with this invention include but are not limited to thepromoters selected from the group consisting of: the SCSV3, SCSV4,SCSV5, and SCSV7 promoters (Schunmann et al. (2003) Plant FunctionalBiology 30:453-60; the rolC gene promoter of Agrobacteriumrhizogenes(Kiyokawa et al. (1994) Plant Physiology 104:801-02;Pandolfini et al. (2003) BioMedCentral (BMC) Biotechnology 3:7,(www.biomedcentral.com/1472-6750/3/7); Graham et al. (1997) Plant Mol.Biol. 33:729-35; Guivarc'h et al. (1996); Almon et al. (1997) PlantPhysiol. 115:1599-607; the rolA gene promoter of Agrobacteriumrhizogenes (Dehio et al. (1993) Plant Mol. Biol. 23:1199-210); thepromoter of the Agrobacterium tumefaciens T-DNA gene 5 (Korber et al.(1991) EMBO J. 10:3983-91); the rice sucrose synthase RSs1 gene promoter(Shi et al. (1994) J. Exp. Bot. 45:623-31); the CoYMV or Commelinayellow mottle badnavirus promoter (Medberry et al. (1992) Plant Cell4:185-92; Zhou et al. (1998) Chin. J. Biotechnol. 14:9-16); the CFDV orcoconut foliar decay virus promoter (Rohde et al. (1994) Plant Mol.Biol. 27:623-28; Hehn and Rhode (1998) J. Gen. Virol. 79:1495-99); theRTBV or rice tungro bacilliform virus promoter (Yin and Beachy (1995)Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80); the peaglutamin synthase GS3A gene (Edwards et al. (1990) Proc. Natl. Acad.Sci. USA 87:3459-63; Brears et al. (1991) Plant J. 1:235-44); the invCD111 and inv CD141 promoters of the potato invertase genes (Hedley etal. (2000) J. Exp. Botany 51:817-21); the promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991) Proc. Natl. Acad. Sci. USA 88:5212-16); theVAHOXI promoter region (Tornero et al. (1996) Plant J. 9:639-48); thepea cell wall invertase gene promoter (Zhang et al. (1996) PlantPhysiol. 112:1111-17); the promoter of the endogenous cotton proteinrelated to chitinase of US published patent application 20030106097, anacid invertase gene promoter from carrot (Ramloch-Lorenz et al. (1993)The Plant J. 4:545-54); the promoter of the sulfate transporter gene,Sultr1; 3 (Yoshimoto et al. (2003) Plant Physiol. 131:1511-17); apromoter of a sucrose synthase gene (Nolte and Koch (1993) PlantPhysiol. 101:899-905); and the promoter of a tobacco sucrose transportergene (Kuhn et al. (1997) Science 275-1298-1300).

Possible promoters also include the Black Cherry promoter for PrunasinHydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797,859), Thioredoxin Hpromoter from cucumber and rice (Fukuda A et al. (2005). Plant CellPhysiol. 46(11):1779-86), Rice (RSs1) (Shi, T. Wang et al. (1994). J.Exp. Bot. 45(274): 623-631) and maize sucrose synthase-1 promoters(Yang., N-S. et al. (1990) PNAS 87:4144-4148), PP2 promoter from pumpkinGuo, H. et al. (2004) Transgenic Research 13:559-566), At SUC2 promoter(Truernit, E. et al. (1995) Planta 196(3):564-70., At SAM-1(S-adenosylmethionine synthetase) (Mijnsbrugge K V. et al. (1996) PlantCell. Physiol. 37(8): 1108-1115), and the Rice tungro bacilliform virus(RTBV) promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J.4(1):71-79).

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. The above list of selectable marker genes is not meant tobe limiting. Any selectable marker gene can be used with thecompositions and methods described herein.

VI. Compositions Comprising Silencing Elements

One or more of the polynucleotides comprising the silencing element maybe provided as an external composition such as a spray or powder to theplant, plant part, seed, a plant insect pest, or an area of cultivation.In another example, a plant is transformed with a DNA construct orexpression cassette for expression of at least one silencing element. Ineither composition, the silencing element, when ingested by an insect,can reduce the level of a target pest sequence and thereby control thepest (i.e., a Coleopteran plant pest including a Diabrotica plant pest,such as, D. virgifera virgifera, D. barberi, D. virgifera zeae, D.speciosa, or D. undecimpunctata howardi). It is recognized that thecomposition may comprise a cell (such as plant cell or a bacterialcell), in which a polynucleotide encoding the silencing element isstably incorporated into the genome and operably linked to promotersactive in the cell. Compositions comprising a mixture of cells, somecells expressing at least one silencing element are also encompassed. Inother embodiments, compositions comprising the silencing elements arenot contained in a cell. In such embodiments, the composition can beapplied to an area inhabited by a plant insect pest. In one embodiment,the composition is applied externally to a plant (i.e., by spraying afield or area of cultivation) to protect the plant from the pest.Methods of applying nucleotides in such a manner are known to those ofskill in the art.

A composition disclosed herein may further be formulated as bait. Inthis embodiment, the compositions comprise a food substance or anattractant which enhances the attractiveness of the composition to thepest.

A composition comprising the silencing element may be formulated in anagriculturally suitable and/or environmentally acceptable carrier. Suchcarriers may be any material that the animal, plant or environment to betreated can tolerate. Furthermore, the carrier must be such that thecomposition remains effective at controlling a plant insect pest.Examples of such carriers include water, saline, Ringer's solution,dextrose or other sugar solutions, Hank's solution, and other aqueousphysiologically balanced salt solutions, phosphate buffer, bicarbonatebuffer and Tris buffer. In addition, the composition may includecompounds that increase the half-life of a composition. Variousinsecticidal formulations can also be found in, for example, USPublications 2008/0275115, 2008/0242174, 2008/0027143, 2005/0042245, and2004/0127520.

It is recognized that the polynucleotides comprising sequences encodingthe silencing element may be used to transform organisms to provide forhost organism production of these components, and subsequent applicationof the host organism to the environment of the target pest(s). Such hostorganisms include baculoviruses, bacteria, and the like. In this manner,the combination of polynucleotides encoding the silencing element may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may be stablyincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

Microbial hosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the sequences encoding the silencing element, and desirably, providefor improved protection of the components from environmental degradationand inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir, and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing the polynucleotidecomprising the silencing element into the microbial host underconditions that allow for stable maintenance and expression of suchnucleotide encoding sequences. For example, expression cassettes can beconstructed which include the nucleotide constructs of interest operablylinked with the transcriptional and translational regulatory signals forexpression of the nucleotide constructs, and a nucleotide sequencehomologous with a sequence in the host organism, whereby integrationwill occur, and/or a replication system that is functional in the host,whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EP 0480762A2;Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3^(rd)edition; Cold Spring Harbor Laboratory Press, Plainview, N.Y.); Davis etal. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.); and the references cited therein.

Suitable host cells include the prokaryotes and the lower eukaryotes,such as fungi. Illustrative prokaryotes, both Gram-negative andGram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia,Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia,Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceaeand Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetesand Ascomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell mayinclude ease of introducing the coding sequence into the host,availability of expression systems, efficiency of expression, stabilityin the host, and the presence of auxiliary genetic capabilities.Characteristics of interest for use as a pesticide microcapsule includeprotective qualities, such as thick cell walls, pigmentation, andintracellular packaging or formation of inclusion bodies; leaf affinity;lack of mammalian toxicity; attractiveness to pests for ingestion; andthe like. Other considerations include ease of formulation and handling,economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.,phylloplane organisms such as Pseudomonas spp., Erwinia spp., andFlavobacterium spp., and other such organisms, including Pseudomonasaeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillusthuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The sequences encoding the silencing elements encompassed by theinvention may be introduced into microorganisms that multiply on plants(epiphytes) to deliver these components to potential target pests.Epiphytes, for example, can be gram-positive or gram-negative bacteria.

A silencing element may be fermented in a bacterial host and theresulting bacteria processed and used as a microbial spray in the samemanner that Bacillus thuringiensis strains have been used asinsecticidal sprays. Any suitable microorganism can be used for thispurpose. By way of example, Pseudomonas has been used to expressBacillus thuringiensis endotoxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide Gaertner et al.(1993), in Advanced Engineered Pesticides, ed. L. Kim (Marcel Decker,Inc.).

Alternatively, the components of the invention are produced byintroducing heterologous genes into a cellular host. Expression of theheterologous sequences results, directly or indirectly, in theintracellular production of a silencing element. These compositions maythen be formulated in accordance with conventional techniques forapplication to the environment hosting a target pest, e.g., soil, water,and foliage of plants. See, for example, EPA 0192319, and the referencescited therein.

A transformed microorganism can be formulated with an acceptable carrierinto separate or combined compositions that are, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, and an emulsifiable concentrate, an aerosol,an impregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients (i.e., at least one silencing element) are normally appliedin the form of compositions and can be applied to the crop area, plant,or seed to be treated. For example, the compositions may be applied tograin in preparation for or during storage in a grain bin or silo, etc.The compositions may be applied simultaneously or in succession withother compounds. Methods of applying an active ingredient or acomposition that contains at least one silencing element include, butare not limited to, foliar application, seed coating, and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono- or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions comprising a silencing element may be in a suitableform for direct application or as a concentrate of primary compositionthat requires dilution with a suitable quantity of water or otherdilutant before application.

The compositions (including the transformed microorganisms) may beapplied to the environment of an insect pest (such as a Coleoptera plantpest or a Diabrotica plant pest) by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pests as a protective measure. For example, thecomposition(s) and/or transformed microorganism(s) may be mixed withgrain to protect the grain during storage. It is generally important toobtain good control of pests in the early stages of plant growth, asthis is the time when the plant can be most severely damaged. Thecompositions can conveniently contain another insecticide if this isthought necessary. In an embodiment of the invention, the composition(s)is applied directly to the soil, at a time of planting, in granular formof a composition of a carrier and dead cells of a Bacillus strain ortransformed microorganism of the invention. Another embodiment is agranular form of a composition comprising an agrochemical such as, forexample, an herbicide, an insecticide, a fertilizer, in an inertcarrier, and dead cells of a Bacillus strain or transformedmicroorganism of the invention.

VII. Plants, Plant Parts, and Methods of Introducing Sequences intoPlants

In one embodiment, the methods of the invention involve introducing apolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide in such a manner that thesequence gains access to the interior of a cell of the plant. Themethods of the invention do not depend on a particular method forintroducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotides into plants are known inthe art including, but not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec transformation (WO 00/28058). Also see Weissinger etal. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens).

In certain embodiments, a silencing element disclosed herein may beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the protein or variants or fragments thereofdirectly into the plant or the introduction of the transcript into theplant. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet.202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al.(1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) TheJournal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, polynucleotides can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector systems and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Such methods include the use of particles coated withpolyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotides disclosed herein may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. Further, it is recognized that promoters may also encompasspromoters utilized for transcription by viral RNA polymerases. Methodsfor introducing polynucleotides into plants and expressing a proteinencoded therein, involving viral DNA or RNA molecules, are known in theart. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology5:209-221.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853. Briefly, the polynucleotides disclosed herein may becontained in transfer cassette flanked by two non-recombinogenicrecombination sites. The transfer cassette is introduced into a planthaving stably incorporated into its genome a target site which isflanked by two non-recombinogenic recombination sites that correspond tothe sites of the transfer cassette. An appropriate recombinase isprovided and the transfer cassette is integrated at the target site. Thepolynucleotide of interest is thereby integrated at a specificchromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the compositions and methods described herein providetransformed seeds (also referred to as “transgenic seed”) having apolynucleotide disclosed herein, for example, an expression cassette,stably incorporated into their genome.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The compositions and methods described herein may be used fortransformation of any plant species, including, but not limited to,monocots and dicots. Examples of plant species of interest include, butare not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the compositions and methodsdescribed herein include, for example, pines such as loblolly pine(Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Incertain embodiments, the compositions and methods described herein canbe used with plants such as crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsand sugarcane plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

VIII. Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more targetpolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orvariants or fragments thereof, or complements thereof, as disclosedherein with one or more additional polynucleotides resulting in theproduction or suppression of multiple polypeptide sequences. Transgenicplants comprising stacks of polynucleotide sequences can be obtained byeither or both of traditional breeding methods or through geneticengineering methods. These methods include, but are not limited to,breeding individual lines each comprising a polynucleotide of interest,transforming a transgenic plant comprising an expression constructcomprising various target polynucleotides as set forth in SEQ ID NOS.:1-53 or 107-254, or encoding silencing elements directed to such targetsequence variants or fragments thereof, or complements thereof, asdisclosed herein with a subsequent gene and co-transformation of genesinto a single plant cell. As used herein, the term “stacked” includeshaving the multiple traits present in the same plant (i.e., both traitsare incorporated into the nuclear genome, one trait is incorporated intothe nuclear genome and one trait is incorporated into the genome of aplastid or both traits are incorporated into the genome of a plastid).In one non-limiting example, “stacked traits” comprise a molecular stackwhere the sequences are physically adjacent to each other. A trait, asused herein, refers to the phenotype derived from a particular sequenceor groups of sequences. Co-transformation of polynucleotides can becarried out using single transformation vectors comprising multiplepolynucleotides or polynucleotides carried separately on multiplevectors. If the sequences are stacked by genetically transforming theplants, the polynucleotide sequences of interest can be combined at anytime and in any order. The traits can be introduced simultaneously in aco-transformation protocol with the polynucleotides of interest providedby any combination of transformation cassettes. For example, if twosequences will be introduced, the two sequences can be contained inseparate transformation cassettes (trans) or contained on the sametransformation cassette (cis). Expression of the sequences can be drivenby the same promoter or by different promoters. It is further recognizedthat polynucleotide sequences can be stacked at a desired genomiclocation using a site-specific recombination system. See, for example,WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO1999/25853.

In some embodiments the various target polynucleotides as set forth inSEQ ID NOS.: 1-53 or 107-254, silencing elements directed to such targetsequences, and variants or fragments thereof, or complements thereof, asdisclosed herein, alone or stacked with one or more additional insectresistance traits can be stacked with one or more additional inputtraits (e.g., herbicide resistance, fungal resistance, virus resistance,stress tolerance, disease resistance, male sterility, stalk strength,and the like) or output traits (e.g., increased yield, modifiedstarches, improved oil profile, balanced amino acids, high lysine ormethionine, increased digestibility, improved fiber quality, droughtresistance, and the like). Thus, the polynucleotide embodiments can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests.

Transgenes useful for stacking include, but are not limited to, to thoseas described herein below.

i. Transgenes that Confer Resistance to Insects or Disease

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang,et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007)Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins fromPhotorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) TheOpen Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied andEnvir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; aPIP-1 polypeptide of US Patent Publication US20140007292; an AfiP-1Aand/or AfIP-1B polypeptide of US Patent Publication US20140033361; aPHI-4 polypeptide of US Patent Publication US20140274885 andUS20160040184; a PIP-47 polypeptide of PCT Publication NumberWO2015/023846, a PIP-72 polypeptide of PCT Publication NumberWO2015/038734; a PtIP-50 polypeptide and a PtIP-65 polypeptide of PCTPublication Number WO2015/120270; a PtIP-83 polypeptide of PCTPublication Number WO2015/120276; a PtIP-96 polypeptide of PCT SerialNumber PCT/US15/55502; and 6-endotoxins including, but not limited to,the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 and Cry55classes of 6-endotoxin genes and the B. thuringiensis cytolytic Cyt1 andCyt2 genes. Members of these classes of B. thuringiensis insecticidalproteins include, but are not limited to Cry1Aa1 (Accession #AAA22353);Cry1Aa2 (Accession #Accession #AAA22552); Cry1Aa3 (Accession #BAA00257);Cry1Aa4 (Accession #CAA31886); Cry1Aa5 (Accession #BAA04468); Cry1Aa6(Accession #AAA86265); Cry1Aa7 (Accession #AAD46139); Cry1Aa8 (Accession#126149); Cry1Aa9 (Accession #BAA77213); Cry1Aa10 (Accession #AAD55382);Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13(Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15(Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17(Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19(Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21(Accession #JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1(Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession#AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession#CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession#CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession#CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #I12419);Cry1Ab12 (Accession #AAC64003); Cry1Ab13 (Accession #AAN76494); Cry1Ab14(Accession #AAG16877); Cry1Ab15 (Accession #AAO13302); Cry1Ab16(Accession #AAK55546); Cry1Ab17 (Accession #AAT46415); Cry1Ab18(Accession #AAQ88259); Cry1Ab19 (Accession #AAW31761); Cry1Ab20(Accession #ABB72460); Cry1Ab21 (Accession #ABS18384); Cry1Ab22(Accession #ABW87320); Cry1Ab23 (Accession #HQ439777); Cry1Ab24(Accession #HQ439778); Cry1Ab25 (Accession #HQ685122); Cry1Ab26(Accession #HQ847729); Cry1Ab27 (Accession #JN135249); Cry1Ab28(Accession #JN135250); Cry1Ab29 (Accession #JN135251); Cry1Ab30(Accession #JN135252); Cry1Ab31 (Accession #JN135253); Cry1Ab32(Accession #JN135254); Cry1Ab33 (Accession #AAS93798); Cry1Ab34(Accession #KC156668); Cry1Ab-like (Accession #AAK14336); Cry1Ab-like(Accession #AAK14337); Cry1Ab-like (Accession #AAK14338); Cry1Ab-like(Accession #ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession#AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession#AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession#AAA86266); Cry1Ac7 (Accession #AAB46989); Cry1Ac8 (Accession#AAC44841); Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession#CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession#I12418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession#AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession#AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession#AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession#ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession#ABZ01836); Cry1Ac23 (Accession #CAQ30431); Cry1Ac24 (Accession#ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession#FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession#ACM90319); Cry1Ac29 (Accession #DQ438941); Cry1Ac30 (Accession#GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession#HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession#HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession#JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession#AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1Ae1 (Accession#AAA22410); Cry1Af1 (Accession #AAB82749); Cry1Ag1 (Accession#AAD46137); Cry1Ah1 (Accession #AAQ14326); Cry1Ah2 (Accession#ABB76664); Cry1Ah3 (Accession #HQ439779); Cry1A11 (Accession#AA039719); Cry1A12 (Accession #HQ439780); Cry1A-like (Accession#AAK14339); Cry1Ba1 (Accession #CAA29898); Cry1Ba2 (Accession#CAA65003); Cry1Ba3 (Accession #AAK63251); Cry1Ba4 (Accession#AAK51084); Cry1Ba5 (Accession #AB020894); Cry1Ba6 (Accession#ABL60921); Cry1Ba7 (Accession #HQ439781); Cry1Bb1 (Accession#AAA22344); Cry1Bb2 (Accession #HQ439782); Cry1Bc1 (Accession#CAA86568); Cry1Bd1 (Accession #AAD10292); Cry1Bd2 (Accession#AAM93496); Cry1Be1 (Accession #AAC32850); Cry1Be2 (Accession#AAQ52387); Cry1Be3 (Accession #ACV96720); Cry1Be4 (Accession#HM070026); Cry1Bf1 (Accession #CAC50778); Cry1Bf2 (Accession#AAQ52380); Cry1Bg1 (Accession #AA039720); Cry1Bh1 (Accession#HQ589331); Cry1Bi1 (Accession #KC156700); Cry1Ca1 (Accession#CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3 (Accession#AAA22343); Cry1Ca4 (Accession #CAA01886); Cry1Ca5 (Accession#CAA65457); Cry1Ca6 [1](Accession #AAF37224); Cry1Ca7 (Accession#AAG50438); Cry1Ca8 (Accession #AAM00264); Cry1Ca9 (Accession#AAL79362); Cry1Ca10 (Accession #AAN16462); Cry1Ca11 (Accession#AAX53094); Cry1Ca12 (Accession #HM070027); Cry1Ca13 (Accession#HQ412621); Cry1Ca14 (Accession #JN651493); Cry1Cb1 (Accession #M97880);Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession #ACD50894);Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession #CAA38099);Cry1Da2 (Accession #I76415); Cry1Da3 (Accession #HQ439784); Cry1db1(Accession #CAA80234); Cry1db2 (Accession #AAK48937); Cry1Dc1 (Accession#ABK35074); Cry1Ea1 (Accession #CAA37933); Cry1Ea2 (Accession#CAA39609); Cry1Ea3 (Accession #AAA22345); Cry1Ea4 (Accession#AAD04732); Cry1Ea5 (Accession #A15535); Cry1Ea6 (Accession #AAL50330);Cry1Ea7 (Accession #AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9(Accession #HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11(Accession #JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession#AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession#HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession#CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession#AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession#AAO13295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession#ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession#CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession#AAO13756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession#CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession#HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession#CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession#AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession#CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession#AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession#AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession#CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession#ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession#FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession#GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession#HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession#JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession#JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession#JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession#JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession#JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession#JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession#JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession#ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession#HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession#ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession#JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession#JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession#AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession#AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession#AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession#KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession#AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession#AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession#AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession#JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession#AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession#CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession#HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession#HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession#KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession#KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession#AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession #D86064);Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession #CAA10671); Cry2Aa6(Accession #CAA10672); Cry2Aa7 (Accession #CAA10670); Cry2Aa8 (Accession#AAO13734); Cry2Aa9 (Accession #AA013750); Cry2Aa10 (Accession#AAQ04263); Cry2Aa11 (Accession #AAQ52384); Cry2Aa12 (Accession#ABI83671); Cry2Aa13 (Accession #ABL01536); Cry2Aa14 (Accession#ACF04939); Cry2Aa15 (Accession #JN426947); Cry2Ab1 (Accession#AAA22342); Cry2Ab2 (Accession #CAA39075); Cry2Ab3 (Accession#AAG36762); Cry2Ab4 (Accession #AA013296); Cry2Ab5 (Accession#AAQ04609); Cry2Ab6 (Accession #AAP59457); Cry2Ab7 (Accession#AAZ66347); Cry2Ab8 (Accession #ABC95996); Cry2Ab9 (Accession#ABC74968); Cry2Ab10 (Accession #EF157306); Cry2Ab11 (Accession#CAM84575); Cry2Ab12 (Accession #ABM21764); Cry2Ab13 (Accession#ACG76120); Cry2Ab14 (Accession #ACG76121); Cry2Ab15 (Accession#HM037126); Cry2Ab16 (Accession #GQ866914); Cry2Ab17 (Accession#HQ439789); Cry2Ab18 (Accession #JN135255); Cry2Ab19 (Accession#JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession#JN135258); Cry2Ab22 (Accession #JN135259); Cry2Ab23 (Accession#JN135260); Cry2Ab24 (Accession #JN135261); Cry2Ab25 (Accession#JN415485); Cry2Ab26 (Accession #JN426946); Cry2Ab27 (Accession#JN415764); Cry2Ab28 (Accession #JN651494); Cry2Ac1 (Accession#CAA40536); Cry2Ac2 (Accession #AAG35410); Cry2Ac3 (Accession#AAQ52385); Cry2Ac4 (Accession #ABC95997); Cry2Ac5 (Accession#ABC74969); Cry2Ac6 (Accession #ABC74793); Cry2Ac7 (Accession#CAL18690); Cry2Ac8 (Accession #CAM09325); Cry2Ac9 (Accession#CAM09326); Cry2Ac10 (Accession #ABN15104); Cry2Ac11 (Accession#CAM83895); Cry2Ac12 (Accession #CAM83896); Cry2Ad1 (Accession#AAF09583); Cry2Ad2 (Accession #ABC86927); Cry2Ad3 (Accession#CAK29504); Cry2Ad4 (Accession #CAM32331); Cry2Ad5 (Accession#CA078739); Cry2Ae1 (Accession #AAQ52362); Cry2Af1 (Accession#AB030519); Cry2Af2 (Accession #GQ866915); Cry2Ag1 (Accession#ACH91610); Cry2Ah1 (Accession #EU939453); Cry2Ah2 (Accession#ACL80665); Cry2Ah3 (Accession #GU073380); Cry2Ah4 (Accession#KC156702); Cry2A11 (Accession #FJ788388); Cry2Aj (Accession #); Cry2Ak1(Accession #KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession#AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession#CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession#AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession#CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession#AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession#AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession#CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession#JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession#AAA74198); Cry3Bb3 (Accession #I15475); Cry3Ca1 (Accession #CAA42469);Cry4Aa1 (Accession #CAA68485); Cry4Aa2 (Accession #BAA00179); Cry4Aa3(Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like(Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession#CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession#BAA00178); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession#ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession#FJ403208); Cry4Cb2 (Accession #FJ597622); Cry4Cc1 (Accession#FJ403207); Cry5Aa1 (Accession #AAA67694); Cry5Ab1 (Accession#AAA67693); Cry5Ac1 (Accession #I34543); Cry5Ad1 (Accession #ABQ82087);Cry5Ba1 (Accession #AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3(Accession #AFJ04417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession#ZP_04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession#ZP_04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession#ZP_04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession#AAM46849); Cry6Aa3 (Accession #ABH03377); Cry6Ba1 (Accession#AAA22358); Cry7Aa1 (Accession #AAA22351); Cry7Ab1 (Accession#AAA21120); Cry7Ab2 (Accession #AAA21121); Cry7Ab3 (Accession#ABX24522); Cry7Ab4 (Accession #EU380678); Cry7Ab5 (Accession#ABX79555); Cry7Ab6 (Accession #ACI44005); Cry7Ab7 (Accession#ADB89216); Cry7Ab8 (Accession #GU145299); Cry7Ab9 (Accession#ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession#KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession#KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession#HM572236); Cry7Da3 (Accession #KC156679); Cry7Ea1 (Accession#HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession#EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession#EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession#KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession#KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession#KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession#KC156651); Cry7Ia1 (Accession #KC156665); Cry7Ja1 (Accession#KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession#BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession#AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession#KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession#AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession#CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession#AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession#ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession#BD133574); Cry8Da3 (Accession #BD133575); Cry8db1 (Accession#BAF93483); Cry8Ea1 (Accession #AAQ73470); Cry8Ea2 (Accession#EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession#AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession#AFH78109); Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession#ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession#AAW81032); Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession#GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession#KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession#KC156677); Cry8Ja1 (Accession #EU625348); Cry8Ka1 (Accession#FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession#HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession#GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession#EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession#HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession#HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession#AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession#KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession#ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession#CAA41425); Cry9Aa3 (Accession #GQ249293); Cry9Aa4 (Accession#GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession#AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession#GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession#CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession#BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession#GQ249293); Cry9Da4 (Accession #GQ249297); Cry9db1 (Accession#AAX78439); Cry9Dc1 (Accession #KC156683); Cry9Ea1 (Accession#BAA34908); Cry9Ea2 (Accession #AAO12908); Cry9Ea3 (Accession#ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession#ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession#FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession#JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession#GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession#AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession#GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession#KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession#AAC63366); Cry10Aa1 (Accession #AAA22614); Cry10Aa2 (Accession#E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession#AFB18318); Cry10A-like (Accession #DQ167578); Cry11Aa1 (Accession#AAA22352); Cry11Aa2 (Accession #AAA22611); Cry11Aa3 (Accession#CAD30081); Cry11Aa4 (Accession #AFB18319); Cry11Aa-like (Accession#DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11Bb1 (Accession#AAC97162); Cry11Bb2 (Accession #HM068615); Cry12Aa1 (Accession#AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession#AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession#AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession#CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession#AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession#CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession#AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession#ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession#GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession #I66477);Cry21Ba1 (Accession #BAC06484); Cry21Ca1 (Accession #JF521577); Cry21Ca2(Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1(Accession #I34547); Cry22Aa2 (Accession #CAD43579); Cry22Aa3 (Accession#ACD93211); Cry22Ab1 (Accession #AAK50456); Cry22Ab2 (Accession#CAD43577); Cry22Ba1 (Accession #CAD43578); Cry22Bb1 (Accession#KC156672); Cry23Aa1 (Accession #AAF76375); Cry24Aa1 (Accession#AAC61891); Cry24Ba1 (Accession #BAD32657); Cry24Ca1 (Accession#CAJ43600); Cry25Aa1 (Accession #AAC61892); Cry26Aa1 (Accession#AAD25075); Cry27Aa1 (Accession #BAA82796); Cry28Aa1 (Accession#AAD24189); Cry28Aa2 (Accession #AAG00235); Cry29Aa1 (Accession#CAC80985); Cry30Aa1 (Accession #CAC80986); Cry30Ba1 (Accession#BAD00052); Cry30Ca1 (Accession #BAD67157); Cry30Ca2 (Accession#ACU24781); Cry30Da1 (Accession #EF095955); Cry30db1 (Accession#BAE80088); Cry30Ea1 (Accession #ACC95445); Cry30Ea2 (Accession#FJ499389); Cry30Fa1 (Accession #AC122625); Cry30Ga1 (Accession#ACG60020); Cry30Ga2 (Accession #HQ638217); Cry31Aa1 (Accession#BAB11757); Cry31Aa2 (Accession #AAL87458); Cry31Aa3 (Accession#BAE79808); Cry31Aa4 (Accession #BAF32571); Cry31Aa5 (Accession#BAF32572); Cry31Aa6 (Accession #BAI44026); Cry31Ab1 (Accession#BAE79809); Cry31Ab2 (Accession #BAF32570); Cry31Ac1 (Accession#BAF34368); Cry31Ac2 (Accession #AB731600); Cry31Ad1 (Accession#BAI44022); Cry32Aa1 (Accession #AAG36711); Cry32Aa2 (Accession#GU063849); Cry32Ab1 (Accession #GU063850); Cry32Ba1 (Accession#BAB78601); Cry32Ca1 (Accession #BAB78602); Cry32Cb1 (Accession#KC156708); Cry32Da1 (Accession #BAB78603); Cry32Ea1 (Accession#GU324274); Cry32Ea2 (Accession #KC156686); Cry32Eb1 (Accession#KC156663); Cry32Fa1 (Accession #KC156656); Cry32Ga1 (Accession#KC156657); Cry32Ha1 (Accession #KC156661); Cry32Hb1 (Accession#KC156666); Cry32Ia1 (Accession #KC156667); Cry32Ja1 (Accession#KC156685); Cry32Ka1 (Accession #KC156688); Cry32La1 (Accession#KC156689); Cry32Ma1 (Accession #KC156690); Cry32Mb1 (Accession#KC156704); Cry32Na1 (Accession #KC156691); Cry32Oa1 (Accession#KC156703); Cry32Pa1 (Accession #KC156705); Cry32Qa1 (Accession#KC156706); Cry32Ra1 (Accession #KC156707); Cry32Sa1 (Accession#KC156709); Cry32Ta1 (Accession #KC156710); Cry32Ua1 (Accession#KC156655); Cry33Aa1 (Accession #AAL26871); Cry34Aa1 (Accession#AAG50341); Cry34Aa2 (Accession #AAK64560); Cry34Aa3 (Accession#AAT29032); Cry34Aa4 (Accession #AAT29030); Cry34Ab1 (Accession#AAG41671); Cry34Ac1 (Accession #AAG50118); Cry34Ac2 (Accession#AAK64562); Cry34Ac3 (Accession #AAT29029); Cry34Ba1 (Accession#AAK64565); Cry34Ba2 (Accession #AAT29033); Cry34Ba3 (Accession#AAT29031); Cry35Aa1 (Accession #AAG50342); Cry35Aa2 (Accession#AAK64561); Cry35Aa3 (Accession #AAT29028); Cry35Aa4 (Accession#AAT29025); Cry35Ab1 (Accession #AAG41672); Cry35Ab2 (Accession#AAK64563); Cry35Ab3 (Accession #AY536891); Cry35Ac1 (Accession#AAG50117); Cry35Ba1 (Accession #AAK64566); Cry35Ba2 (Accession#AAT29027); Cry35Ba3 (Accession #AAT29026); Cry36Aa1 (Accession#AAK64558); Cry37Aa1 (Accession #AAF76376); Cry38Aa1 (Accession#AAK64559); Cry39Aa1 (Accession #BAB72016); Cry40Aa1 (Accession#BAB72018); Cry40Ba1 (Accession #BAC77648); Cry40Ca1 (Accession#EU381045); Cry40Da1 (Accession #ACF15199); Cry41Aa1 (Accession#BAD35157); Cry41Ab1 (Accession #BAD35163); Cry41Ba1 (Accession#HM461871); Cry41Ba2 (Accession #ZP_04099652); Cry42Aa1 (Accession#BAD35166); Cry43Aa1 (Accession #BAD15301); Cry43Aa2 (Accession#BAD95474); Cry43Ba1 (Accession #BAD15303); Cry43Ca1 (Accession#KC156676); Cry43Cb1 (Accession #KC156695); Cry43Cc1 (Accession#KC156696); Cry43-like (Accession #BAD15305); Cry44Aa (Accession#BAD08532); Cry45Aa (Accession #BAD22577); Cry46Aa (Accession#BAC79010); Cry46Aa2 (Accession #BAG68906); Cry46Ab (Accession#BAD35170); Cry47Aa (Accession #AAY24695); Cry48Aa (Accession#CAJ18351); Cry48Aa2 (Accession #CAJ86545); Cry48Aa3 (Accession#CAJ86546); Cry48Ab (Accession #CAJ86548); Cry48Ab2 (Accession#CAJ86549); Cry49Aa (Accession #CAH56541); Cry49Aa2 (Accession#CAJ86541); Cry49Aa3 (Accession #CAJ86543); Cry49Aa4 (Accession#CAJ86544); Cry49Ab1 (Accession #CAJ86542); Cry50Aa1 (Accession#BAE86999); Cry50Ba1 (Accession #GU446675); Cry50Ba2 (Accession#GU446676); Cry51Aa1 (Accession #ABI14444); Cry51Aa2 (Accession#GU570697); Cry52Aa1 (Accession #EF613489); Cry52Ba1 (Accession#FJ361760); Cry53Aa1 (Accession #EF633476); Cry53Ab1 (Accession#FJ361759); Cry54Aa1 (Accession #ACA52194); Cry54Aa2 (Accession#GQ140349); Cry54Ba1 (Accession #GU446677); Cry55Aa1 (Accession#ABW88932); Cry54Ab1 (Accession #JQ916908); Cry55Aa2 (Accession#AAE33526); Cry56Aa1 (Accession #ACU57499); Cry56Aa2 (Accession#GQ483512); Cry56Aa3 (Accession #JX025567); Cry57Aa1 (Accession#ANC87261); Cry58Aa1 (Accession #ANC87260); Cry59Ba1 (Accession#JN790647); Cry59Aa1 (Accession #ACR43758); Cry60Aa1 (Accession#ACU24782); Cry60Aa2 (Accession #EA057254); Cry60Aa3 (Accession#EEM99278); Cry60Ba1 (Accession #GU810818); Cry60Ba2 (Accession#EA057253); Cry60Ba3 (Accession #EEM99279); Cry61Aa1 (Accession#HM035087); Cry61Aa2 (Accession #HM132125); Cry61Aa3 (Accession#EEM19308); Cry62Aa1 (Accession #HM054509); Cry63Aa1 (Accession#BAI44028); Cry64Aa1 (Accession #BAJ05397); Cry65Aa1 (Accession#HM461868); Cry65Aa2 (Accession #ZP_04123838); Cry66Aa1 (Accession#HM485581); Cry66Aa2 (Accession #ZP_04099945); Cry67Aa1 (Accession#HM485582); Cry67Aa2 (Accession #ZP_04148882); Cry68Aa1 (Accession#HQ113114); Cry69Aa1 (Accession #HQ401006); Cry69Aa2 (Accession#JQ821388); Cry69Ab1 (Accession #JN209957); Cry70Aa1 (Accession#JN646781); Cry70Ba1 (Accession #ADO51070); Cry70Bb1 (Accession#EEL67276); Cry71Aa1 (Accession #JX025568); Cry72Aa1 (Accession#JX025569).

Examples of 6-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cryproteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8,304,605, Cry1Bof U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins (US PatentApplication Publication Number 2010/0017914); a Cry4 protein; a Cry5protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736,7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008)Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, aCry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; TIC1100, TIC 860, a TIC867, a TIC868, TIC869, and TIC836 ofUS Patent Publication Number 2016/0108428. AXMI-027, AXMI-036, andAXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040,AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 ofWO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585;AXMI-008 of US 2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917;AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457;AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXM1226, AXM1227,AXM1228, AXM1229, AXMI230, and AXMI231 of WO11/103247; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211;AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543;and Cry proteins such as Cry1A and Cry3A having modified proteolyticsites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxinprotein from Bacillus thuringiensis strain VBTS 2528 of US PatentApplication Publication Number 2011/0064710, and an IP1B of PCTpublication number WO 2016/061197. Other Cry proteins are well known toone skilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on theworld-wide web using the “www” prefix). The insecticidal activity of Cryproteins is well known to one skilled in the art (for review, see, vanFrannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE &Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605)); Cry34Ab/35Ab and Cry6Aa(US20130167269); Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry3A andCry1Ab or Vip3Aa (US20130116170); and Cry1F, Cry34Ab1, and Cry35Ab1(PCT/US2010/060818). Pesticidal proteins also include insecticidallipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, andcholesterol oxidases such as from Streptomyces (Purcell et al. (1993)Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins alsoinclude VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos.5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020,and the like. Other VIP proteins are well known to one skilled in theart (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which canbe accessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) PI. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Reg locus that may beutilized as a single locus conversion.

(X) Some embodiments relate to down-regulation of expression of targetgenes in insect pest species by interfering ribonucleic acid (RNA)molecules. PCT Publication WO 2007/074405 describes methods ofinhibiting expression of target genes in invertebrate pests includingColorado potato beetle. PCT Publication WO 2005/110068 describes methodsof inhibiting expression of target genes in invertebrate pests includingin particular Western corn rootworm as a means to control insectinfestation. Furthermore, PCT Publication WO 2009/091864 describescompositions and methods for the suppression of target genes from insectpest species including pests from the Lygus genus.

Nucleic acid molecules including silencing elements for targeting thevacuolar ATPase H subunit, useful for controlling a coleopteran pestpopulation and infestation as described in US Patent ApplicationPublication 2012/0198586. PCT Publication WO 2012/055982 describesribonucleic acid (RNA or double stranded RNA) that inhibits or downregulates the expression of a target gene that encodes: an insectribosomal protein such as the ribosomal protein L19, the ribosomalprotein L40 or the ribosomal protein S27A; an insect proteasome subunitsuch as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasomebeta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomerof the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomerprotein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2A protein which is a putative transmembrane domain protein; an insectprotein belonging to the actin family such as Actin 5C; an insectubiquitin-5E protein; an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport; an insectcrinkled protein which is an unconventional myosin which is involved inmotor activity; an insect crooked neck protein which is involved in theregulation of nuclear alternative mRNA splicing; an insect vacuolarH+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-bindingprotein. PCT publication WO 2007/035650 describes ribonucleic acid (RNAor double stranded RNA) that inhibits or down regulates the expressionof a target gene that encodes Snf7. US Patent Application publication2011/0054007 describes polynucleotide silencing elements targetingRPS10. US Patent Application publication 2014/0275208 and US2015/0257389describe polynucleotide silencing elements targeting RyanR and PAT3. PCTpublications WO 2016/060911, WO 2016/060912, WO 2016/060913, and WO2016/060914 describe polynucleotide silencing elements targeting COPIcoatomer subunit nucleic acid molecules that confer resistance toColeopteran and Hemipteran pests. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describe potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence.

ii. Transgenes that Confer Resistance to a Herbicide.

(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582.

Glyphosate resistance is also imparted to plants that express a geneencoding a glyphosate oxido-reductase enzyme as described more fully inU.S. Pat. Nos. 5,776,760 and 5,463,175. In addition glyphosateresistance can be imparted to plants by the over expression of genesencoding glyphosate N-acetyltransferase. See, for example, U.S. Pat.Nos. 7,462,481; 7,405,074 and US Patent Application Publication NumberUS 2008/0234130. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC Accession Number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. EPApplication Number 0 333 033 to Kumada, et al., and U.S. Pat. No.4,975,374 to Goodman, et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 B1 and 5,879,903. Exemplary genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall, et al., (1992) Theor. Appl. Genet. 83:435. (C) Apolynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC Accession Numbers 53435,67441 and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes, et al., (1992) Biochem. J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluoroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance.

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance.

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

iii. Transgenes that Confer or Contribute to an Altered GrainCharacteristic

(A) Altered fatty acids, for example, by (1) Down-regulation ofstearoyl-ACP to increase stearic acid content of the plant. See,Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO1999/64579 (Genes to Alter Lipid Profiles in Corn); (2) Elevating oleicacid via FAD-2 gene modification and/or decreasing linolenic acid viaFAD-3 gene modification (see, U.S. Pat. Nos. 6,063,947; 6,323,392;6,372,965 and WO 1993/11245); (3) Altering conjugated linolenic orlinoleic acid content, such as in WO 2001/12800; (4) Altering LEC1, AGP,Dek1, Superal1, mi1ps, various Ipa genes such as Ipa1, Ipa3, hpt orhggt. For example, see, WO 2002/42424, WO 1998/22604, WO 2003/011015, WO2002/057439, WO 2003/011015, U.S. Pat. Nos. 6,423,886, 6,197,561,6,825,397 and US Patent Application Publication Numbers US 2003/0079247,US 2003/0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci.92:5620-5624; (5) Genes encoding delta-8 desaturase for makinglong-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and8,338,152), delta-9 desaturase for lowering saturated fats (U.S. Pat.No. 8,063,269), Primula delta 6-desaturase for improving omega-3 fattyacid profiles; (6) Isolated nucleic acids and proteins associated withlipid and sugar metabolism regulation, in particular, lipid metabolismprotein (LMP) used in methods of producing transgenic plants andmodulating levels of seed storage compounds including lipids, fattyacids, starches or seed storage proteins and use in methods ofmodulating the seed size, seed number, seed weights, root length andleaf size of plants (EP 2404499); (7) Altering expression of aHigh-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plantto increase or decrease expression of HSI2 in the plant. Increasingexpression of HSI2 increases oil content while decreasing expression ofHSI2 decreases abscisic acid sensitivity and/or increases droughtresistance (US Patent Application Publication Number 2012/0066794); (8)Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particularly to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904); and (9) Nucleicacid molecules encoding wrinkled1-like polypeptides for modulating sugarmetabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the (1) introduction ofa phytase-encoding gene would enhance breakdown of phytate, adding morefree phosphate to the transformed plant. For example, see, VanHartingsveldt, et al., (1993) Gene 127:87, for a disclosure of thenucleotide sequence of an Aspergillus niger phytase gene; and (2)modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648. which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Sogaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

iv. Genes that Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed. Non-limiting examples include: (A) Introduction of adeacetylase gene under the control of a tapetum-specific promoter andwith the application of the chemical N-Ac-PPT (WO 2001/29237); (B)Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957); and (C) Introduction of the barnase and the barstar gene(Paul, et al., (1992) Plant Mol. Biol. 19:611-622). For additionalexamples of nuclear male and female sterility systems and genes, seealso, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369; 5,824,524;5,850,014 and 6,265,640.

v. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821. Other systems that may be used include the Glnrecombinase of phage Mu (Maeser, et al., (1991) Vicki Chandler, TheMaize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase of E.coli (Enomoto, et al., 1983) and the R/RS system of the pSRi plasmid(Araki, et al., 1992).

vi. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress. Non-limiting examples include: (A) For example, see: WO2000/73475 where water use efficiency is altered through alteration ofmalate; U.S. Pat. Nos. 5,892,009, 5,965,705, 5,929,305, 5,891,859,6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO 2000/060089,WO 2001/026459, WO 2001/035725, WO 2001/034726, WO 2001/035727, WO2001/036444, WO 2001/036597, WO 2001/036598, WO 2002/015675, WO2002/017430, WO 2002/077185, WO 2002/079403, WO 2003/013227, WO2003/013228, WO 2003/014327, WO 2004/031349, WO 2004/076638, WO199809521; (B) WO 199938977 describing genes, including CBF genes andtranscription factors effective in mitigating the negative effects offreezing, high salinity and drought on plants, as well as conferringother positive effects on plant phenotype; (C) US Patent ApplicationPublication Number 2004/0148654 and WO 2001/36596 where abscisic acid isaltered in plants resulting in improved plant phenotype such asincreased yield and/or increased tolerance to abiotic stress; (D) WO2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and 6,992,237where cytokinin expression is modified resulting in plants withincreased stress tolerance, such as drought tolerance, and/or increasedyield. Also see, WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S.Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. No. 6,177,275 and U.S. Pat.No. 6,107,547 (enhancement of nitrogen utilization and altered nitrogenresponsiveness); (E) For ethylene alteration, see, US Patent ApplicationPublication Number 2004/0128719, US Patent Application PublicationNumber 2003/0166197 and WO 2000/32761; (F) For plant transcriptionfactors or transcriptional regulators of abiotic stress, see, e.g., USPatent Application Publication Number 2004/0098764 or US PatentApplication Publication Number 2004/0078852; (G) Genes that increaseexpression of vacuolar pyrophosphatase such as AVP1 (U.S. Pat. No.8,058,515) for increased yield; nucleic acid encoding a HSFA4 or a HSFA5(Heat Shock Factor of the class A4 or A5) polypeptides, an oligopeptidetransporter protein (OPT4-like) polypeptide; a plastochron2-like(PLA2-like) polypeptide or a Wuschel related homeobox 1-like (WOX1-like)polypeptide (U. Patent Application Publication Number US 2011/0283420);(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor; (I) Polynucleotide encoding DTP21polypeptides for conferring drought resistance (US Patent ApplicationPublication Number US 2011/0277181); (J) Nucleotide sequences encodingACC Synthase 3 (ACS3) proteins for modulating development, modulatingresponse to stress, and modulating stress tolerance (US PatentApplication Publication Number US 2010/0287669); (K) Polynucleotidesthat encode proteins that confer a drought tolerance phenotype (DTP) forconferring drought resistance (WO 2012/058528); (L) Tocopherol cyclase(TC) genes for conferring drought and salt tolerance (US PatentApplication Publication Number 2012/0272352); (M) CAAX amino terminalfamily proteins for stress tolerance (U.S. Pat. No. 8,338,661); (N)Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633); (O) Expression of a nucleic acid sequence encodinga polypeptide selected from the group consisting of: GRF polypeptide,RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTPpolypeptide increasing yield-related traits (US Patent ApplicationPublication Number 2011/0061133); and (P) Modulating expression in aplant of a nucleic acid encoding a Class III Trehalose PhosphatePhosphatase (TPP) polypeptide for enhancing yield-related traits inplants, particularly increasing seed yield (US Patent ApplicationPublication Number 2010/0024067).

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

vii. Genes that Confer Increased Yield

Non-limiting examples of genes that confer increased yield are: (A) Atransgenic crop plant transformed by a 1-AminoCyclopropane-1-CarboxylateDeaminase-like Polypeptide (ACCDP) coding nucleic acid, whereinexpression of the nucleic acid sequence in the crop plant results in theplant's increased root growth, and/or increased yield, and/or increasedtolerance to environmental stress as compared to a wild type variety ofthe plant (U.S. Pat. No. 8,097,769); (B) Over-expression of maize zincfinger protein gene (Zm-ZFP1) using a seed preferred promoter has beenshown to enhance plant growth, increase kernel number and total kernelweight per plant (US Patent Application Publication Number2012/0079623); (C) Constitutive over-expression of maize lateral organboundaries (LOB) domain protein (Zm-LOBDP1) has been shown to increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079622); (D) Enhancing yield-related traits inplants by modulating expression in a plant of a nucleic acid encoding aVIM1 (Variant in Methylation 1)-like polypeptide or a VTC2-like(GDP-L-galactose phosphorylase) polypeptide or a DUF1685 polypeptide oran ARF6-like (Auxin Responsive Factor) polypeptide (WO 2012/038893); (E)Modulating expression in a plant of a nucleic acid encoding a Ste20-likepolypeptide or a homologue thereof gives plants having increased yieldrelative to control plants (EP 2431472); and (F) Genes encodingnucleoside diphosphatase kinase (NDK) polypeptides and homologs thereoffor modifying the plant's root architecture (US Patent ApplicationPublication Number 2009/0064373).

IX. Methods of Use

Methods disclosed herein comprise methods for controlling a plant insectpest, such as a Coleopteran, Hemiptera, or Lepidopteran plant pest,including a Diabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan,Bemisia, Halyomorpha, Nezara, or Spodoptera plant pest. In oneembodiment, the method comprises feeding or applying to a plant insectpest a composition comprising a silencing element disclosed herein,wherein said silencing element, when ingested or contacted by a plantinsect pest (i.e., but not limited to, a Coleopteran plant pestincluding a Diabrotica plant pest, such as, D. virgifera virgifera, D.barberi, D. virgifera zeae, D. speciosa, or D. undecimpunctata howardi),reduces the level of a target polynucleotide of the pest and therebycontrols the pest. The pest can be fed the silencing element in avariety of ways. The silencing element may be fed to male, female, orboth sexes of a pest. For example, in an embodiment, a polynucleotideencoding a silencing element, i.e., a silencing element targeting one ormore polynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, isintroduced into a plant. As the plant pest feeds on the plant or partthereof expressing these sequences, the silencing element is deliveredto the pest at larval, adult, or at any or all developmental stages. Inone embodiment, the methods and compositions described herein furthercomprise a transgenic plant comprising a silencing element disclosedherein, wherein the silencing element has sterilization activity atlarval, adult or at any or all developmental stages. When the silencingelement is delivered to the plant in this manner, it is recognized thatthe silencing element can be expressed constitutively or alternatively,it may be produced in a stage-specific manner by employing the variousinducible or tissue-preferred or developmentally regulated promotersthat are discussed elsewhere herein. In certain embodiments, thesilencing element is expressed in the roots, stalk or stem, leafincluding pedicel, xylem and phloem, fruit or reproductive tissue, silk,flowers and all parts therein or any combination thereof. Sterileinsects may result from exposure to silencing elements in this mannerand hence sterilize insects of opposite the sex through competitivemating or SIT.

In another method, a composition comprising at least one silencingelement disclosed herein is applied to a plant. In such embodiments, thesilencing element may be formulated in an agronomically suitable and/orenvironmentally acceptable carrier, which is preferably, suitable fordispersal in fields. In some embodiments, silencing elements targetingdifferent insect stages, pathways, and sexes may be combined forsterility and insecticidal activities. In one embodiment, the silencingelements disclosed herein may be mixed with pesticidal chemicals by tankmix. In addition, the carrier may also include compounds that increasethe half-life of the composition. In certain embodiments, thecomposition comprising the silencing element is formulated in such amanner such that it persists in the environment for a length of timesufficient to allow it to be delivered to a plant insect pest. In suchembodiments, the composition can be applied to an area inhabited by aplant insect pest. In one embodiment, the composition is appliedexternally to a plant (i.e., by spraying a field) to protect the plantfrom pests. Sterile insects that result from exposure to silencingelements may sterilize insects of opposite sex through competitivemating or SIT.

In certain embodiments, the disclosed polynucleotides or constructs canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired trait. A trait, as used herein,refers to the phenotype derived from a particular sequence or groups ofsequences. For example, the polynucleotides described herein may bestacked with any other polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, such as other Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986)Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825,pentin (described in U.S. Pat. No. 5,981,722), and the like. Thecombinations generated may also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides described hereincan also be stacked with any other gene or combination of genes toproduce plants with a variety of desired trait combinations including,but not limited to, traits desirable for animal feed such as high oilgenes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)).

Disclosed polynucleotides can also be stacked with traits desirable fordisease or herbicide resistance (e.g., fumonisin detoxification genes(U.S. Pat. No. 5,792,931); avirulence and disease resistance genes(Jones et al. (1994) Science 266:789; Martin et al. (1993) Science262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase(ALS) mutants that lead to herbicide resistance such as the S4 and/orHra mutations; inhibitors of glutamine synthase such as phosphinothricinor basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)); andtraits desirable for processing or process products such as high oil(e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combinethe polynucleotides with polynucleotides providing agronomic traits suchas male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,drought resistance (e.g., U.S. Pat. No. 7,786,353), flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821).

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants (i.e., molecular stacks), thepolynucleotide sequences of interest can be combined at any time and inany order. For example, a transgenic plant comprising one or moredesired traits can be used as the target to introduce further traits bysubsequent transformation. The traits can be introduced simultaneouslyin a co-transformation protocol with the polynucleotides of interestprovided by any combination of transformation cassettes. For example, iftwo sequences will be introduced, the two sequences can be contained inseparate transformation cassettes (trans) or contained on the sametransformation cassette (cis). Expression of the sequences can be drivenby the same promoter or by different promoters. In certain cases, it maybe desirable to introduce a transformation cassette that will suppressthe expression of the polynucleotide of interest. This may be combinedwith any combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853.

X. Insect Resistance Management Methods

Methods disclosed herein comprise methods for controlling a plant insectpest, such as a Coleopteran, Hemiptera, or Lepidopteran plant pest,including a Diabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan,Bemisia, Halyomorpha, Nezara, or Spodoptera plant pest, such as insectresistance management. Insect resistance management (IRM) is the termused to describe practices aimed at reducing the potential for insectpests to become resistant to a pesticide. Maintenance of Bt (or otherpesticidal protein, chemical, or biological) IRM is of great importancebecause of the threat insect resistance poses to the future use of Btplant-incorporated protectants and Bt technology as a whole. SpecificIRM strategies, such as the high dose/structured refuge strategy, delayinsect resistance to specific Bt proteins produced in corn, cotton, andpotatoes. However, such strategies result in portions of crops beingleft susceptible to one or more pests in order to ensure thatnon-resistant insects develop and become available to mate with anyresistant pests produced in protected crops. Accordingly, from afarmer/producer's perspective, it is highly desirable to have as small arefuge as possible and yet still manage insect resistance, in order thatthe greatest yield be obtained while still maintaining the efficacy ofthe pest control method used, whether Bt, chemical, some other method,or combinations thereof.

An often used IRM strategy is the planting of a refuge (a portion of thetotal acreage using non-Bt/pesticidal trait seed), as it iscommonly-believed that this will delay the development of insectresistance to pesticidal traits by maintaining insect susceptibility.The theoretical basis of the refuge strategy for delaying resistancehinges on the assumption that the frequency and recessiveness of insectresistance is inversely proportional to pest susceptibility; resistancewill be rare and recessive only when pests are very susceptible to thetoxin, and conversely resistance will be more frequent and lessrecessive when pests are not very susceptible. Furthermore, the strategyassumes that resistance to Bt is recessive and is conferred by a singlelocus with two alleles resulting in three genotypes: susceptiblehomozygotes (SS), heterozygotes (RS), and resistant homozygotes (RR). Italso assumes that there will be a low initial resistance allelefrequency and that there will be extensive random mating betweenresistant and susceptible adults. Under ideal circumstances, only rareRR individuals will survive a pesticidal toxin produced by the crop.Both SS and RS individuals will be susceptible to the pesticidal toxin.A structured refuge is a non-Bt/pesticidal trait portion of a grower'sfield or set of fields that provides for the production of susceptible(SS) insects that may randomly mate with rare resistant (RR) insectssurviving the pesticidal trait crop, which may be a Bt trait crop, toproduce susceptible RS heterozygotes that will be killed by theBt/pesticidal trait crop. An integrated refuge is a certain portion ofrandomly planted non-Bt/pesticidal trait portion of a grower's field orset of fields that provides for the production of susceptible (SS)insects that may randomly mate with rare resistant (RR) insectssurviving the pesticidal trait crop to produce susceptible RSheterozygotes that will be killed by the pesticidal trait crop Eachrefuge strategy will remove resistant (R) alleles from the insectpopulations and delay the evolution of resistance.

Another strategy to reduce the need for refuge is the pyramiding oftraits with different modes of action against a target insect pest. Forexample, Bt toxins that have different modes of action stacked in onetransgenic plant are able to have reduced refuge requirements. Differentmodes of action in a stacked combination also maintains the durabilityof each trait, as resistance is slower to develop to each trait.

Currently, the size, placement, and management of the refuge are oftenconsidered critical to the success of refuge strategies to mitigateinsect resistance to the Bt/pesticidal trait produced in corn, cotton,soybean, and other crops. Because of the decrease in yield in refugeplanting areas, some farmers choose to eschew the refuge requirements,and others do not follow the size and/or placement requirements. Theseissues result in either no refuge or less effective refuge, and acorresponding risk of the increase in the development of resistancepests.

Accordingly, there remains a need for methods for managing pestresistance in a plot of pest resistant crop plants. It would be usefulto provide an improved method for the protection of plants, especiallycorn or other crop plants, from feeding damage by pests. It would beparticularly useful if such a method would reduce the requiredapplication rate of conventional chemical pesticides, and also if itwould limit the number of separate field operations that were requiredfor crop planting and cultivation. In addition, it would be useful tohave a method of deploying a transgenic refuge that eliminates theabove-described problems with regard to compliance that dilute or removethe efficacy of many resistance management strategies.

One embodiment relates to a method of reducing the development ofresistant pests comprising providing a plant protection composition to aplant (Bt toxin, transgenic insecticidal protein, other insecticidalproteins, chemical insecticides, insecticidal biologicalentomopathogens, etc.) and contacting the plant pest with a silencingelement, i.e., of a silencing element targeting one or morepolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, whereinthe silencing element, i.e., of a silencing element targeting one ormore polynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254,produces a decrease in expression of one or more of the sequences in thetarget pest and controls the pest and pest population by insectsterilization or SIT.

A further embodiment relates to a method of increasing the durability ofplant pest compositions comprising providing a plant protectioncomposition to a plant (Bt toxin, transgenic insecticidal protein, otherinsecticidal proteins, chemical insecticides, insecticidal biologicalentomopathogens etc.) and contacting a plant pest with the sterilizationsilencing element, i.e., of one or more polynucleotides as set forth inSEQ ID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotides, produces a decrease in expression of one or more of thesequences in the target pest and controls the pest and pest populationby insect sterilization or sterile insect technique. In anotherembodiment, the refuge planted as a strip, a block, or integrated withthe trait seed comprises a plant further comprising a sterilizationsilencing element (for example, a silencing element targeting one ormore polynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254).

In a still further embodiment, the refuge required may be reduced oreliminated by the presence of a sterilization silencing element appliedto the non-refuge plants. In another embodiment, the refuge ornon-refuge may include a silencing element, i.e., of one or morepolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, an expression construct comprising a sequence asset forth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, orsilencing elements targeting said polynucleotides, as a spray, bait,lure, or as a different transgenic plant.

In a further embodiment, a pest insect is feed a diet comprising one ormore polynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, an expression construct comprising a sequence asset forth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, orsilencing elements targeting said polynucleotides, and said insects arereleased onto plants at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days followingfeeding. In a still further embodiment, the pest insect is a female pestinsect. In a yet further embodiment, the pest insect is a pest insect,and the pest insect is fed during a larval or adult stage. Insectsterilization may result from male or female sterility, mating ofsterile insects, reduction of sperm count, egg production or viability.

In certain embodiments, the compositions and methods disclosed herein,targeting a sterile gene via RNAi technology, and stacking apolynucleotide encoding a silencing element disclosed herein with aninsecticidal protein in a transgenic plant may provide effective controlof Coleoptera and potentially extend the durability of Coleopteraninsecticidal traits. The extended durability may be a consequence ofminimizing the transmission of resistance alleles from Coleopteranbeetles that were able to complete their developmental life cycle whilefeeding on transgenic roots expressing a stack of an insecticidalprotein(s) and a RNAi sterility trait disclosed herein.

Current IRM strategy requires a high dose of Bt toxins to minimizeinsect resistance development. Due to phyto-toxicity, it can bedifficult to achieve the required high dose. Integrated pest management(IPM) by different means of insect control may be used to delay insectresistance exposed to a sub-optimal dose of protein toxin, such as a Bttoxin. RNAi mediated SIT may be deployed as part of an IPM strategy.

As used herein, the term “pesticidal” is used to refer to a toxic effectagainst a pest (e.g., CRW), and includes activity of either, or both, anexternally supplied pesticide and/or an agent that is produced by thecrop plants. As used herein, the term “different mode of pesticidalaction” includes the pesticidal effects of one or more resistancetraits, whether introduced into the crop plants by transformation ortraditional breeding methods, such as binding of a pesticidal toxinproduced by the crop plants to different binding sites (i.e., differenttoxin receptors and/or different sites on the same toxin receptor) inthe gut membranes of corn rootworms or through RNA interference.

XI. Application Methods

In one embodiment, one or more polynucleotides as set forth in SEQ IDNOS.: 1-53 or 107-254, or complements thereof, an expression constructcomprising a sequence as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, or silencing elements targeting said polynucleotidesequences, and compositions comprising said sequences can be applieddirectly to the seed. For example, one or more polynucleotides as setforth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, anexpression construct comprising a sequence as set forth in SEQ ID NOS.:1-53 or 107-254, or complements thereof, or silencing elements targetingsaid polynucleotide sequences, used in the compositions and methodsdisclosed herein can be applied without additional components andwithout having been diluted.

In one embodiment, sprays, baits, lures, attractants, and seedtreatments can comprise one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequences.

In another embodiment, one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequences areapplied to the seed in the form of a suitable formulation. Suitableformulations and methods for the treatment of seed are known to theperson skilled in the art and are described, for example, in thefollowing documents: U.S. Pat. Nos. 4,272,417 A, 4,245,432 A, 4,808,430A, 5,876,739 A, US 2003/0176428 A1, WO 2002/080675 A1, WO 2002/028186A2.

The one or more polynucleotides as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, an expression construct comprising asequence as set forth in SEQ ID NOS.: 1-53 or 107-254, or complementsthereof, or silencing elements targeting said polynucleotide sequences,and compositions comprising said sequences can be converted intocustomary seed dressing formulations, such as solutions, emulsions,suspensions, powders, foams, slurries or other coating materials forseed, and also ULV formulations. These formulations are prepared in aknown manner by mixing the one or more polynucleotides as set forth inSEQ ID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequenceswith customary additives, such as, for example, customary extenders andalso solvents or diluents, colorants, wetting agents, dispersants,emulsifiers, defoamers, preservatives, secondary thickeners, adhesives,gibberellins and water as well.

In another embodiment, suitable colorants that may be present in theseed dressing formulations include all colorants customary for suchpurposes. Use may be made both of pigments, of sparing solubility inwater, and of dyes, which are soluble in water. Examples that may bementioned include the colorants known under the designations RhodamineB, C.I. Pigment Red 112, and C.I. Solvent Red 1

In another embodiment, suitable wetting agents that may be present inthe seed dressing formulations include all substances that promotewetting and are customary in the formulation of active agrochemicalsubstances. With preference it is possible to usealkylnaphthalene-sulphonates, such as diisopropyl- ordiisobutylnaphthalene-sulphonates.

In still another embodiment, suitable dispersants and/or emulsifiersthat may be present in the seed dressing formulations include allnonionic, anionic, and cationic dispersants that are customary in theformulation of active agrochemical substances. In one embodiment,nonionic or anionic dispersants or mixtures of nonionic or anionicdispersants can be used. In one embodiment, nonionic dispersants includebut are not limited to ethylene oxide-propylene oxide block polymers,alkylphenol polyglycol ethers, and tristyrylphenol polyglycol ethers,and their phosphated or sulphated derivatives.

In still another embodiment, defoamers that may be present in the seeddressing formulations to be used according to the invention include allfoam-inhibiting compounds that are customary in the formulation ofagrochemically active compounds including, but not limited, to siliconedefoamers, magnesium stearate, silicone emulsions, long-chain alcohols,fatty acids and their salts and also organofluorine compounds andmixtures thereof.

In still another embodiment, secondary thickeners that may be present inthe seed dressing formulations include all compounds which can be usedfor such purposes in agrochemical compositions, including but notlimited to cellulose derivatives, acrylic acid derivatives,polysaccharides, such as xanthan gum or Veegum, modified clays,phyllosilicates, such as attapulgite and bentonite, and also finelydivided silicic acids.

Suitable adhesives that may be present in the seed dressing formulationsto be used according to the invention include all customary binderswhich can be used in seed dressings. Polyvinylpyrrolidone, polyvinylacetate, polyvinyl alcohol and tylose may be mentioned as beingpreferred.

In another embodiment, one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or complements thereof, or an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequences isapplied to soil in a first application step, applied to seed in a secondapplication, and to applied to the foliar region of a plant in a thirdapplication.

As used herein, applying one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or a complement thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or a complement thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequences toa seed, a plant, or plant part includes contacting the seed, plant, orplant part directly and/or indirectly with the one or morepolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, an expression construct comprising a sequence asset forth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, orsilencing elements targeting said polynucleotide sequences, andcompositions comprising said sequences. In one embodiment, one or morepolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, an expression construct comprising a sequence asset forth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, orsilencing elements targeting said polynucleotide sequences, andcompositions comprising said sequences can be directly applied as aspray, a rinse, or a powder, or any combination thereof.

In another aspect, one or more polynucleotides as set forth in SEQ IDNOS.: 1-53 or 107-254, or complements thereof, an expression constructcomprising a sequence as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, or silencing elements targeting said polynucleotidesequences, and compositions comprising said sequences can be applieddirectly to a plant or plant part as a powder. As used herein, a powderis a dry or nearly dry bulk solid composed of a large number of veryfine particles that may flow freely when shaken or tilted. A dry ornearly dry powder composition disclosed herein preferably contains a lowpercentage of water, such as, for example, in various aspects, less than5%, less than 2.5%, or less than 1% by weight.

In a further embodiment, one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, may be introduced in a bacteria, a yeast, orfungus by transformation techniques known to the skilled artisan, andsaid transformed bacteria, yeast, or fungus applied to a plant, soilthat the plant is growing in, to a hydroponic medium, seed, or anyapplied per any of the foregoing application methods as described hereinabove.

In one embodiment, the one or more polynucleotides as set forth in SEQID NOS.: 1-53 or 107-254, or complements thereof, an expressionconstruct comprising a sequence as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, or silencing elements targeting saidpolynucleotide sequences, and compositions comprising said sequences maybe formulated by encapsulation technology to improve stability. In oneembodiment the encapsulation technology may comprise a bead polymer fortimed release over time. In one embodiment, the encapsulated one or morepolynucleotides as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, an expression construct comprising a sequence asset forth in SEQ ID NOS.: 1-53 or 107-254, or complements thereof, orsilencing elements targeting said polynucleotide sequences, andcompositions comprising said sequences may be applied in a separateapplication of beads in-furrow to the seeds. In another embodiment, theencapsulated one or more polynucleotides as set forth in SEQ ID NOS.:1-53 or 107-254, or complements thereof, an expression constructcomprising a sequence as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, or silencing elements targeting said polynucleotidesequences, and compositions comprising said sequences may be co-appliedalong with seeds simultaneously.

The coating agent usable for the sustained release microparticles of anencapsulation embodiment may be a substance which is useful for coatingthe microgranular form with the substance to be supported thereon. Anycoating agent which can form a coating difficultly permeable for thesupported substance may be used in general, without any particularlimitation. For example, higher saturated fatty acid, wax, thermoplasticresin, thermosetting resin and the like may be used.

Examples of useful higher saturated fatty acid include stearic acid,zinc stearate, stearic acid amide and ethylenebis-stearic acid amide;those of wax include synthetic waxes such as polyethylene wax, carbonwax, Hoechst wax, and fatty acid ester; natural waxes such as carnaubawax, bees wax and Japan wax; and petroleum waxes such as paraffin waxand petrolatum. Examples of thermoplastic resin include polyolefins suchas polyethylene, polypropylene, polybutene and polystyrene; vinylpolymers such as polyvinyl acetate, polyvinyl chloride, polyvinylidenechloride, polyacrylic acid, polymethacrylic acid, polyacrylate andpolymethacrylate; diene polymers such as butadiene polymer, isoprenepolymer, chloroprene polymer, butadiene-styrene copolymer,ethylene-propylene-diene copolymer, styrene-isoprene copolymer,MMA-butadiene copolymer and acrylonitrile-butadiene copolymer;polyolefin copolymers such as ethylene-propylene copolymer,butene-ethylene copolymer, butene-propylene copolymer, ethylene-vinylacetate copolymer, ethylene-acrylic acid copolymer, styreneacrylic acidcopolymer, ethylene-methacrylic acid copolymer, ethylene-methacrylicester copolymer, ethylene-carbon monoxide copolymer, ethylene-vinylacetate-carbon monoxide copolymer, ethylene-vinyl acetate-vinyl chloridecopolymer and ethylene-vinyl acetate-acrylic copolymer; and vinylchloride copolymers such as vinyl chloride-vinyl acetate copolymer andvinylidene chloride-vinyl chloride copolymer. Examples of thermosettingresin include polyurethane resin, epoxy resin, alkyd resin, unsaturatedpolyester resin, phenolic resin, urea-melamine resin, urea resin andsilicone resin. Of those, thermoplastic acrylic ester resin,butadienestyrene copolymer resin, thermosetting polyurethane resin andepoxy resin are preferred, and among the preferred resins, particularlythermosetting polyurethane resin is preferred. These coating agents canbe used either singly or in combination of two or more kinds.

In one embodiment, one or more polynucleotides as set forth in SEQ IDNOS.: 1-53 or 107-254, or complements thereof, an expression constructcomprising a sequence as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, or silencing elements targeting said polynucleotidesequences, and compositions comprising said sequences can be formulatedto further comprise an entomopathogen. The methods and compositions ofthe disclosure, in one embodiment relate to a composition comprising oneor more one or more polynucleotides as set forth in SEQ ID NOS.: 1-53 or107-254, or complements thereof, an expression construct comprising asequence as set forth in SEQ ID NOS.: 1-53 or 107-254, or complementsthereof, or silencing elements targeting said polynucleotide sequences,and compositions comprising said sequences and one or more biocontrolagents. As used herein, the term “biocontrol agent” (“BCA”) includes oneor more bacteria, fungi or yeasts, protozoas, viruses, entomopathogenicnematodes, and botanical extracts, or products produced bymicroorganisms including proteins or secondary metabolite, andinnoculants that have one or both of the following characteristics: (1)inhibits or reduces plant infestation and/or growth of pathogens, pests,or insects, including but not limited to pathogenic fungi, bacteria, andnematodes, as well as arthropod pests such as insects, arachnids,chilopods, diplopods, or that inhibits plant infestation and/or growthof a combination of plant pathogens, pests, or insects; (2) improvesplant performance; (3) improves plant yield; (4) improves plant vigor;and (5) improves plant health.

XII. Knockout of Target Genes Using Cas/CRISPR

In one embodiment, one or more polynucleotides as set forth in SEQ IDNOS.: 1-53 or 107-254, or complements thereof, an expression constructcomprising a sequence as set forth in SEQ ID NOS.: 1-53 or 107-254, orcomplements thereof, or silencing elements targeting saidpolynucleotides, and compositions comprising said sequences, can be canbe introduced into the genome of a plant using genome editingtechnologies, or previously introduced polynucleotides encoding asilencing element disclosed herein in the genome of a plant may beedited using genome editing technologies. For example, the disclosedpolynucleotides can be introduced into a desired location in the genomeof a plant through the use of double-stranded break technologies such asTALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like.For example, the disclosed polynucleotides can be introduced into adesired location in a genome using a CRISPR-Cas system, for the purposeof site-specific insertion. The desired location in a plant genome canbe any desired target site for insertion, such as a genomic regionamenable for breeding or may be a target site located in a genomicwindow with an existing trait of interest. Existing traits of interestcould be either an endogenous trait or a previously introduced trait.

In another aspect, where the disclosed polynucleotide encoding asilencing element has previously been introduced into a genome, genomeediting technologies may be used to alter or modify the introducedpolynucleotide sequence. Site specific modifications that can beintroduced into the disclosed polynucleotide encoding a silencingelement compositions include those produced using any method forintroducing site specific modification, including, but not limited to,through the use of gene repair oligonucleotides (e.g. US Publication2013/0019349), or through the use of double-stranded break technologiessuch as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, andthe like. Such technologies can be used to modify the previouslyintroduced polynucleotide through the insertion, deletion orsubstitution of nucleotides within the introduced polynucleotide.Alternatively, double-stranded break technologies can be used to addadditional nucleotide sequences to the introduced polynucleotide.Additional sequences that may be added include, additional expressionelements, such as enhancer and promoter sequences. In anotherembodiment, genome editing technologies may be used to positionadditional insecticidally-active proteins in close proximity to thedisclosed polynucleotide compositions disclosed herein within the genomeof a plant, in order to generate molecular stacks ofinsecticidally-active proteins. An “altered target site,” “alteredtarget sequence.” “modified target site,” and “modified target sequence”are used interchangeably herein and refer to a target sequence asdisclosed herein that comprises at least one alteration when compared tonon-altered target sequence. Such “alterations” include, for example:(i) replacement of at least one nucleotide, (ii) a deletion of at leastone nucleotide, (iii) an insertion of at least one nucleotide, or (iv)any combination of (i)-(iii).

In one embodiment, the methods comprise creating an insect, or colonythereof, wherein the target gene is edited so that it is no longerfunction, thereby creating a sterile insect. The polynucleotide sequenceof the target gene can be used to knockout the target genepolynucleotide in an insect by means known to those skilled in the art,including, but not limited to TALENs, meganucleases, zinc fingernucleases, CRISPR-Cas, and the like. See Ma et al (2014), ScientificReports, 4: 4489; Daimon et al (2013), Development, Growth, andDifferentiation, 56(1): 14-25; and Eggleston et al (2001) BMC Genetics,2:11. One embodiment comprises an insect with an edited polynucleotideof one or more polynucleotides as set forth in SEQ ID NOS.: 1-53 whereinthe edit produces a decrease in expression of or a nonfunctionalpolypeptide and controls the pest and pest population by insectsterilization and sterile insect technique.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1: Nucleic Acid Sequences

Nucleic acid sequences disclosed herein comprise the following nucleicacid sequences. Certain sequences are exemplary and were shown to haveinsect sterilization activity against corn rootworms using the assaymethods described in Examples 2, 3, and 6 as set forth below. Suchsequences or their complements can be used in the methods as describedherein above and below. Methods for making inhibitory sequences areknown in the art. DNA constructs, vectors, transgenic cells, plants,seeds or products described herein may comprise one or more of thefollowing nucleic acid or amino acid sequences, or a portion of one ormore of the disclosed sequences. Non-limiting examples of targetpolynucleotides are set forth below in Table 1, or variants andfragments thereof, and complements thereof, including, for example, SEQID NOS.: 1-53 or 107-254, and variants and fragments thereof, andcomplements thereof. The list of sequences referred to herein, SEQ IDNOS.: 1-53 and 107-254, is included herein below.

TABLE 1 VgR RNAi target fragments. SEQ ID NO. Species Common nameFragment ID 1 Diabrotica virgifera Western Corn Rootworm Transcript*virgifera 2 Diabrotica virgifera Western Corn Rootworm ORF** virgifera 3Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag1 virgifera 4Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag2 virgifera 5Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag3 virgifera 6Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag4 virgifera 7Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag5 virgifera 8Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag6 virgifera 9Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag7 virgifera 10Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag8 virgifera 11Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag9 virgifera 12Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag10 virgifera 13Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag11 virgifera 14Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag12 virgifera 15Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag13 virgifera 16Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag14 virgifera 17Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag15 virgifera 18Diabrotica virgifera Western Corn Rootworm DV-VGR-Frag16 virgifera 19Diabrotica Southern Corn Rootworm ORF undecimpunctata 20 DiabroticaSouthern Corn Rootworm Transcript undecimpunctata 21 LeptinotarsaColorado Potato Beetle ORF decemlineata 22 Leptinotarsa Colorado PotatoBeetle Transcript decemlineata 23 Phyllotreta striolata Striped FleaBeetle ORF 24 Phyllotreta striolata Striped Flea Beetle Transcript 25Halyomorpha halys Brown Marmorated Stink ORF Bug 26 Halyomorpha halysBrown Marmorated Stink Transcript Bug 27 Acyrthosiphon pisum Pea AphidORF 28 Acyrthosiphon pisum Pea Aphid Transcript 29 Bemisia tabaciSilverleaf Whitefly ORF 30 Bemisia tabaci Silverleaf Whitefly Transcript31 Spodoptera litura Cotton Leafworm ORF 32 Spodoptera litura CottonLeafworm Transcript 33 Phyllotreta cruciferae Crucifer Flea Beetle ORF34 Phyllotreta cruciferae Crucifer Flea Beetle Transcript 35 Nezaraviridula Southern Green Stink Bug ORF 36 Diabrotica virgifera WesternCorn Rootworm DV-CUL3 virgifera 37 Diabrotica virgifera Western CornRootworm DV-NCLB virgifera 38 Diabrotica virgifera Western Corn RootwormDV-MAEL virgifera 39 Diabrotica virgifera Western Corn Rootworm DV-GUDUvirgifera 40 Diabrotica virgifera Western Corn Rootworm DV-GSKTvirgifera 41 Diabrotica virgifera Western Corn Rootworm DV-WTS virgifera42 Diabrotica virgifera Western Corn Rootworm DV-CASP virgifera 43Diabrotica virgifera Western Corn Rootworm DV-CYCA virgifera 44Diabrotica virgifera Western Corn Rootworm DV-CUL3-FRAG1 virgifera 45Diabrotica virgifera Western Corn Rootworm DV-NCLB-FRAG1 virgifera 46Diabrotica virgifera Western Corn Rootworm DV-MAEL-FRAG1 virgifera 47Diabrotica virgifera Western Corn Rootworm DV-GUDU-FRAG1 virgifera 48Diabrotica virgifera Western Corn Rootworm DV-GSKT-FRAG1 virgifera 49Diabrotica virgifera Western Corn Rootworm DV-WTS-FRAG1 virgifera 50Diabrotica virgifera Western Corn Rootworm DV-CASP-FRAG1 virgifera 51Diabrotica virgifera Western Corn Rootworm DV-CYCA-FRAG1 virgifera 52Spodoptera frugiperda Fall Armyworm ORF 53 Spodoptera frugiperda FallArmyworm Transcript 107 Diabrotica virgifera Western Corn RootwormDV-ADE2 virgifera 108 Diabrotica virgifera Western Corn Rootworm DV-HANGvirgifera 109 Diabrotica virgifera Western Corn Rootworm DV-KL3virgifera 110 Diabrotica virgifera Western Corn Rootworm DV-PORINvirgifera 111 Diabrotica virgifera Western Corn Rootworm DV-SU(VAR)205virgifera 112 Diabrotica virgifera Western Corn Rootworm DV-PARKvirgifera 113 Diabrotica virgifera Western Corn Rootworm DV-POEvirgifera 114 Diabrotica virgifera Western Corn Rootworm DV-MBD-LIKEvirgifera 115 Diabrotica virgifera Western Corn Rootworm DV-PGLYM78virgifera 116 Diabrotica virgifera Western Corn Rootworm DV-HIRAvirgifera 117 Diabrotica virgifera Western Corn Rootworm DV-PUFvirgifera 118 Diabrotica virgifera Western Corn Rootworm DV-TUDvirgifera 119 Diabrotica virgifera Western Corn Rootworm DV-FAFvirgifera 120 Diabrotica virgifera Western Corn Rootworm DV-NUP44Avirgifera 121 Diabrotica virgifera Western Corn Rootworm DV-GEKvirgifera 122 Diabrotica virgifera Western Corn Rootworm DV-HTSvirgifera 123 Diabrotica virgifera Western Corn Rootworm DV-CDK7virgifera 124 Diabrotica virgifera Western Corn Rootworm DV-DLG1virgifera 125 Diabrotica virgifera Western Corn Rootworm DV-DM virgifera126 Diabrotica virgifera Western Corn Rootworm DV-EGG virgifera 127Diabrotica virgifera Western Corn Rootworm DV-HRG virgifera 128Diabrotica virgifera Western Corn Rootworm DV-MR virgifera 129Diabrotica virgifera Western Corn Rootworm DV-CG17083 virgifera 130Diabrotica virgifera Western Corn Rootworm DV-CG3565 virgifera 131Diabrotica virgifera Western Corn Rootworm DV-CYCB virgifera 132Diabrotica virgifera Western Corn Rootworm DV-KNRL virgifera 133Diabrotica virgifera Western Corn Rootworm DV-MEI virgifera 134Diabrotica virgifera Western Corn Rootworm DV-TWE virgifera 135Diabrotica virgifera Western Corn Rootworm DV-BOULE virgifera 136Diabrotica virgifera Western Corn Rootworm DV-ADE2-FRAG1 virgifera 137Diabrotica virgifera Western Corn Rootworm DV-HANG-FRAG1 virgifera 138Diabrotica virgifera Western Corn Rootworm DV-KL3-FRAG1 virgifera 139Diabrotica virgifera Western Corn Rootworm DV-PORIN-FRAG1 virgifera 140Diabrotica virgifera Western Corn Rootworm DV-SU(VAR)205-FRAG1 virgifera141 Diabrotica virgifera Western Corn Rootworm DV-PARK-FRAG1 virgifera142 Diabrotica virgifera Western Corn Rootworm DV-POE-FRAG1 virgifera143 Diabrotica virgifera Western Corn Rootworm DV-MBD-LIKE-FRAG1virgifera 144 Diabrotica virgifera Western Corn RootwormDV-PGLYM78-FRAG1 virgifera 145 Diabrotica virgifera Western CornRootworm DV-HIRA-FRAG1 virgifera 146 Diabrotica virgifera Western CornRootworm DV-PUF-FRAG1 virgifera 147 Diabrotica virgifera Western CornRootworm DV-TUD-FRAG1 virgifera 148 Diabrotica virgifera Western CornRootworm DV-FAF-FRAG1 virgifera 149 Diabrotica virgifera Western CornRootworm DV-NUP44A-FRAG1 virgifera 150 Diabrotica virgifera Western CornRootworm DV-GEK-FRAG1 virgifera 151 Diabrotica virgifera Western CornRootworm DV-HTS-FRAG1 virgifera 152 Diabrotica virgifera Western CornRootworm DV-CDK7-FRAG1 virgifera 153 Diabrotica virgifera Western CornRootworm DV-DLG1-FRAG1 virgifera 154 Diabrotica virgifera Western CornRootworm DV-DM-FRAG1 virgifera 155 Diabrotica virgifera Western CornRootworm DV-EGG-FRAG1 virgifera 156 Diabrotica virgifera Western CornRootworm DV-HRG-FRAG1 virgifera 157 Diabrotica virgifera Western CornRootworm DV-MR-FRAG1 virgifera 158 Diabrotica virgifera Western CornRootworm DV-CG17083-FRAG1 virgifera 159 Diabrotica virgifera WesternCorn Rootworm DV-CG3565-FRAG1 virgifera 160 Diabrotica virgifera WesternCorn Rootworm DV-CYCB-FRAG1 virgifera 161 Diabrotica virgifera WesternCorn Rootworm DV-KNRL-FRAG1 virgifera 162 Diabrotica virgifera WesternCorn Rootworm DV-MEI-FRAG1 virgifera 163 Diabrotica virgifera WesternCorn Rootworm DV-TWE-FRAG1 virgifera 164 Diabrotica virgifera WesternCorn Rootworm DV-BOULE-FRAG1 virgifera 165 Diabrotica virgifera WesternCorn Rootworm DV-REPH virgifera 166 Diabrotica virgifera Western CornRootworm DV-ARMI virgifera 167 Diabrotica virgifera Western CornRootworm DV-LOQS virgifera 168 Diabrotica virgifera Western CornRootworm DV-SCNY virgifera 169 Diabrotica virgifera Western CornRootworm DV-AGO3 virgifera 170 Diabrotica virgifera Western CornRootworm DV-DIA virgifera 171 Diabrotica virgifera Western Corn RootwormDV-DNC virgifera 172 Diabrotica virgifera Western Corn Rootworm DV-CHIvirgifera 173 Diabrotica virgifera Western Corn Rootworm DV-SXLvirgifera 174 Diabrotica virgifera Western Corn Rootworm DV-SLGAvirgifera 175 Diabrotica virgifera Western Corn Rootworm DV-PAPLA1virgifera 176 Diabrotica virgifera Western Corn Rootworm DV-REPH-FRAG1virgifera 177 Diabrotica virgifera Western Corn Rootworm DV-ARMI-FRAG1virgifera 178 Diabrotica virgifera Western Corn Rootworm DV-LOQS-FRAG1virgifera 179 Diabrotica virgifera Western Corn Rootworm DV-SCNY-FRAG1virgifera 180 Diabrotica virgifera Western Corn Rootworm DV-AGO3-FRAG1virgifera 181 Diabrotica virgifera Western Corn Rootworm DV-DIA-FRAG1virgifera 182 Diabrotica virgifera Western Corn Rootworm DV-DNC-FRAG1virgifera 183 Diabrotica virgifera Western Corn Rootworm DV-CHI-FRAG1virgifera 184 Diabrotica virgifera Western Corn Rootworm DV-SXL-FRAG1virgifera 185 Diabrotica virgifera Western Corn Rootworm DV-SLGA-FRAG1virgifera 186 Diabrotica virgifera Western Corn Rootworm DV-PAPLA1-FRAG1virgifera 187 Diabrotica Southern Corn Rootworm DU-BOULE undecimpunctata188 Leptinotarsa Colorado Potato Beetle LD-BOULE decemlineata 189Phyllotreta cruciferae Crucifer Flea Beetle PC-BOULE 190 Phyllotretastriolata Striped Flea Beetle PS-BOULE 191 Vibidia duodecimguttata12-Spotted Ladybeetle VD-BOULE 192 Orius insidiosus Insidious Flower BugOI-BOULE 193 Lygus hesperus Western Plant Bug LH-BOULE 194 Megacoptacribraria Kudzu Bug MC-BOULE 195 Euschistus servus Brown Stink BugES-BOULE 196 Nezara viridula Southern Green Stink Bug NV-BOULE 197Helicoverpa zea Corn Earworm HZ-BOULE 198 Ostrinia nubilalis EuropeanCorn Borer ON-BOULE 199 Spodoptera frugiperda Fall Armyworm SF-BOULE 200Diabrotica Southern Corn Rootworm DU-MAEL undecimpunctata 201Leptinotarsa Colorado Potato Beetle LD-MAEL decemlineata 202 Phyllotretastriolata Striped Flea Beetle PS-MAEL 203 Phyllotreta cruciferaeCrucifer Flea Beetle PC-MAEL 204 Epilachna varivestis Mexican BeanBeetle EV-MAEL 205 Tribolium castaneum Red Flour Beetle TC-MAEL 206Vibidia duodecimguttata 12-Spotted Ladybeetle VD-MAEL 207 Helicoverpazea Corn Earworm HZ-MAEL 208 Megacopta cribraria Kudzu Bug MC-MAEL 209Nezara viridula Southern Green Stink Bug NV-MAEL 210 Euschistus servusBrown Stink Bug ES-MAEL 211 Orius insidiosus Insidious Flower BugOI-MAEL 212 Manduca sexta Hornworm MS-MAEL 213 Spodoptera frugiperdaFall Armyworm SF-MAEL 214 Ostrinia nubilalis European Corn Borer ON-MAEL215 Lygus hesperus Western Plant Bug LH-MAEL 216 Pectinophoragossypiella Pink Bollworm PG-MAEL 217 Diabrotica barberi Northern CornRootworm DB-NCLB 218 Diabrotica Southern Corn Rootworm DU-NCLBundecimpunctata 219 Phyllotreta striolata Striped Flea Beetle PS-NCLB220 Phyllotreta cruciferae Crucifer Flea Beetle PC-NCLB 221 LeptinotarsaColorado Potato Beetle LD-NCLB decemlineata 222 Tribolium castaneum RedFlour Beetle TC-NCLB 223 Epilachna varivestis Mexican Bean BeetleEV-NCLB 224 Vibidia duodecimguttata 12-Spotted Ladybeetle VD-NCLB 225Pectinophora gossypiella Pink Bollworm PG-NCLB 226 Spodoptera frugiperdaFall Armyworm SF-NCLB 227 Ostrinia nubilalis European Corn Borer ON-NCLB228 Manduca sexta Hornworm MS-NCLB 229 Helicoverpa zea Corn EarwormHZ-NCLB 230 Megacopta cribraria Kudzu Bug MC-NCLB 231 Nezara viridulaSouthern Green Stink Bug NV-NCLB 232 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG2 virgifera 233 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG3 virgifera 234 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG4 virgifera 235 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG5 virgifera 236 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG6 virgifera 237 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG7 virgifera 238 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG8 virgifera 239 Diabrotica virgifera Western CornRootworm DV-BOULE-FRAG9 virgifera 240 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG2 virgifera 241 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG3 virgifera 242 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG4 virgifera 243 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG5 virgifera 244 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG6 virgifera 245 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG7 virgifera 246 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG8 virgifera 247 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG9 virgifera 248 Diabrotica virgifera Western CornRootworm DV-MAEL-FRAG10 virgifera 249 Diabrotica virgifera Western CornRootworm DV-NCLB-FRAG2 virgifera 250 Diabrotica virgifera Western CornRootworm DV-NCLB-FRAG3 virgifera 251 Diabrotica virgifera Western CornRootworm DV-NCLB-FRAG4 virgifera 252 Diabrotica virgifera Western CornRootworm DV-NCLB-FRAG5 virgifera 253 Diabrotica virgifera Western CornRootworm DV-NCLB-FRAG6 virgifera 254 Diabrotica virgifera Western CornRootworm DV-RPS10 virgifera *“Transcipt” indicates that the sequence isa full-length mRNA transcript sequence. **“ORF” indicates that thesequence corresponds to the open reading frame sequence.

Example 2: Western Corn Rootworm (WCRW) Adult Sterilization by VgR dsRNA

Artificial diet for WCRW adults was prepared using a modified protocol(Rangasamy M et al. (2012). Pest Manag. Sci. 68(4):587-91; andNowatzki™, et al. (2006) J Econ. Entomol. 99(3):927-30). The modifieddiet was designed for use in the diet incorporated bioassay describedherein below (25 μl test sample: 75 μl prepared diet) and was producedusing standard 96-well micro-titer plates. WCRW adults consumedsignificant proportions of the diet within 24 hours, and controlmortality remains <15% during the 2-3 week study period. For all thebioassays described herein, WCRW life stages (adults and larvae) werekept under standard conditions, for example, Jackson, J. J. (1986), pp25-48. In Krysan J. L. and Miller T. A. (eds): Methods for the Study ofpest Diabrotica. Springer-Verlag, New York. 260 pp, and Branson, T. F.,et al. (1988), J. Econ. Entomal. 81(1): 410-414 (1988).

Beetles from the same batch were categorized in to two groups. The firsttest group consisted of male and females of <10 days old (range 2-5 daysold) (hereinafter referred to as “younger females”). The younger femaleswere in their preoviposition period (the period before oviposition ofthe first eggs). The second group consisted of >11 days old and matedfemales (hereinafter referred to as “older females”) and they were inoviposition period (females are ready to lay eggs). For the youngerfemale group 100 beetles (50 females and 50 males) and for the olderfemale group 50 mated females were arranged for each treatment. Thefollowing three treatments were compared 1) sterile DI water (control);2) GFP dsRNA (negative control, GenBank Accession #AY233272.1; SEQ IDNO: 104 herein); and 3) VgR dsRNA fragment 2 (SEQ ID NO: 4). Thebioassay was carried out using a diet incorporation methodology. Testsamples of GFP dsRNA and VgR dsRNA were prepared separately and 25 μl ofthe respective samples were incorporated into 75 μl of modified WCRWadult artificial diet per well in 96-well micro-titer plates for a finalconcentration of 100 ppm. For control 25 μl of sterile DI water wasincorporated into 75 μl of modified WCRW adult artificial diet per well.

The effects on individual WCRW adult beetle were confined usingindividual wells of 32 cell trays (C-D International, Pitman, NJ)provided with single diet pill (mixture of 25 μl test sample and 75 μlof modified WCRW adult artificial diet) for 24 hours. After 24 hour,treated adults were collected, counted and transferred to theirrespective holding cages and provided standard SCRW dry adult diet andwater source until the end of the study period (22-25 days).

WCRW eggs were collected daily for 13-14 days starting from 24 hour or 7days after exposure for the older and younger female group,respectively. Eggs were collected using oviposition dish. Collected eggswere incubated in a heat and humidity controlled growth chamber (25° C.,65%±5% reltaive humidity (RH)) with controlled light/dark cycles (16 hrlight:8 hr darkness) for 12-14 days before processing.

Several small aliquots of egg-agar suspensions were dispensed onto ahatch plate (petri-dish containing 2% water agar and two layers offilter paper) for counting and/or hatch test depending on the number ofeggs obtained for a given day. For the egg hatch test, samples (1-6)each containing 25 μl of egg-agar suspension were dispensed onto thehatch plate as described above and the lids secured with micropore tapeto avoid larval escape. Total number of eggs in each 25 μl sample wascounted prior incubation. Egg hatch plates were then incubated in a heatand humidity controlled growth chamber (25° C., 65%±5% RH) withcontrolled light/dark cycles (16 hr light:8 hr darkness) for three days.Egg hatch was counted over three days period by counting the number ofeggs showing larval emergence hole. For each treatment, four treatedfemale and male beetles (younger female group) and four females (olderfemale group) were sampled for gene suppression at 4 and 8 days afterexposure for the older and younger female group respectively.

For dsRNA in vitro transcript (“IVT”) production, PCR was performedusing target specific forward and reverse primers (see Table 2 below)with a T7 promoter sequence at the 5′ end of each primer. The dsRNAsamples were produced from PCR template using Ambion Megascript HighYield Transcription Kit (Thermo Fisher Scientific, Grand Island, N.Y.).An agarose gel was run to check for yield and product size. For realtime qRTPCR assay, total RNA was extracted with MirVana miRNA IsolationKit, treated by TURBO DNase Kit, assayed by SuperScript® III Platinum®One-Step qRT-PCR Kit with ROX according to manufacturer's instructions(Thermo Fisher Scientific). Relative expression was derived by deltadelta Ct method (Livak, K. J. and T. D. Schmittgen (2001). Methods25(4): 402-408) using WCRW RPS10 as reference (i.e., SEQ ID NO: 8 in US2011/0054007; also SEQ ID NOs.: 102 and 103, ORF and transcript,respectively, herein).

TABLE 2 Primer Sequences IVT Production. Forward Primer Reverse PrimerFragment ID SEQ ID NO. SEQ ID NO. DV-VGR-FRAG1 54 55 DV-VGR-FRAG2 56 57DV-VGR-FRAG3 58 59 DV-VGR-FRAG4 60 61 DV-VGR-FRAG5 62 63 DV-VGR-FRAG6 6465 DV-VGR-FRAG7 66 67 DV-VGR-FRAG8 68 69 DV-VGR-FRAG9 70 71DV-VGR-FRAG10 72 73 DV-VGR-FRAG11 74 75 DV-VGR-FRAG12 76 77DV-VGR-FRAG13 78 79 DV-VGR-FRAG14 80 81 DV-VGR-FRAG15 82 83DV-VGR-FRAG16 84 85 GFP 86 87 GUS 88 89

Data obtained using these methods are shown in FIGS. 1A-1D. Inparticular, FIG. 1A shows the total number of eggs produced within 13-14days by treatment and age group. In the experiment shown in FIG. 1A, theyounger female group contained 50 pairs of male and female beetles,whereas the older female group had 50 mated female beetles. The data inFIG. 1A show that ingestion of the VgR dsRNA significantly reduced thetotal number of eggs produced during the test period. FIG. 1B shows theaverage number of eggs produced per female/day during 13-14 dayoviposition period by treatment and age group. The box plot shows 4quartiles, average, and 95% confidence interval of the mean. The datashow that in both younger and older females, ingestion of the VgR dsRNAreduced the average number of egges produced per day. FIG. 1C shows theeffect of various treatments on overall average egg hatch rate. Datarepresents 13-14 days egg collection period; n=6replication/treatment/day; 5-45 eggs/replication depending on the day(p<0.001). Gene suppression analysis is shown in FIG. 1D for analysiscarried out on WCRW adult beetles 8 days after treatment of female andmale insects for younger age group and 4 days after treatment of femaleinsects for older age group. Relative expression of VgR is shown from 4individual insects for each treatment using WCRW RPS10 gene as referenceand untreated older beetle as normalizer. The box plot shows 4quartiles, average, median, and 95% confidence interval of the mean bytreatment and age group.

Example 3: WCRW Sterilization by Treatment of 3rd Instar Larvae with VgRdsRNA

The effect of treatment of larva on WCRW sterilization by VgR dsRNA (VgRdsRNA fragment 2) was assessed. The study was carried out using 3rdinstar larvae that were harvested from corn mats and acclimatized onstandard WCRW larval diet for 24 h. About 192 larvae were exposed towater and 75 ppm VgR dsRNA fragment 2 (SEQ ID NO: 4) for 1 day using thediet incorporation method described above (25 μl dsRNA and 75 μlartificial WCRW larval diet). Treated larvae were placed in pupationmedium for 15 days. Emerged adults were collected, counted, andtransferred to their respective holding cages and provided standard SCRWdry adult diet with a water source until the end of the study period(22-25 days). Beetle holding cages were kept at room temperature(usually from 22-25° C.) with no RH control. No intentional light/darkcontrol but cages were getting roughly 16:8 Dark and light condition.Beetle holding cages were cleaned maintained twice a week and each timethe beetles received new food and water agar. Adult beetles were keptfor a total of 26 days. Each cage received oviposition dishes after 10days preoviposition period and eggs were collected over a period of for16 days oviposition period, and processed following the method describedabove.

Representative data for this study are shown in FIGS. 2A and 2B. Theaverage total number of eggs produced per female and the average numberof viable eggs produced per female are shown in FIG. 2A. Eggs from 15-42female adult beetles were counted for each indicated treatment. The boxplot of shows 4 quartiles, average, median, and 95% confidence intervalof the mean for each treatment. The data show that for the VgR dsRNAexposed group, the viable egg production remained very low throughoutthe study period. It should be noted that treatment with VgR dsRNA didnot affect adult emergence, and that mortality of adult beetles in theVgR dsRNA group was negligible.

Representative data for VgR gene suppression analysis is shown in FIG.2B. The data were obtained for 10-day old (n=4) and 28-day old (n=15)beetles, which represents day 40 and day 58, respectively, followingtreatment of the 3^(rd) instar larvae. The box plot of relativeexpression by qRTPCR shows 4 quartiles, average, median, and 95%confidence interval of the mean for each treatment in 10 and 28 day oldbeetles. The data were normalized to untreated 3rd instar larvae. Thedata show decreased relative expression of VgR in both age groups.

Example 4: Dose Response of WCRW Sterilization and Gene Suppression byVgR dsRNA Treatment

The dose response effect of dsRNA treatment was determined in youngerand older adult females. The older female group (>11 days old) wascollected and exposed VgR dsRNA using the diet incorporation methodologydescribed above. The treatment groups were exposed for 24 hours. The VgRdsRNA was complementary to SEQ ID NO: 3, and the concentrations testedwere as follows: 0, 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm and 75 ppm. Thetreatment groups consisted of about 40-48 females for each dose level.Egg production was assessed starting 24 hours after exposure andcontinued for 18 days. For each treatment, the total number of femalebeetles used for egg production varies from 40-48 (days 1-6) and 20-28(days 7-18). Eggs were handled and processed following the methodsdescribed above. Six day after exposure 20 treated females wereretrieved from each treatment and were used for gene suppressionanalysis.

The details for each dose treatment group (e.g. number of total eggsanalyzed, number of viable eggs, and net reduction in fecundity) aregiven in FIG. 3A. Eggs were collected and counted over the 18 dayoviposition period. The net reduction in fecundity (NRF) of VgRdsRNA-treated females relative to control (water exposed females) wasestimated using the following formula:

NRF (%)=[1−(NVEt/NVEwc)]*100,

where “NRF” represents the net reduction in fecundity as a percent;“NVEt” repredsents the number of viable eggs in the treatment group; and“NVEwc” represents the number of viable eggs in water (control) treatedgroup. The data show a significant reduction in egg production after 10days of exposure to the VgR dsRNA (see eggs/female day10-18 in FIG. 3A).The data further show that egg production and viability of eggs werenegatively correlated with VgR dsRNA doses. The net reduction infecundity was positively correlated with increased vgR dsRNA doses. FIG.3B shows a box plot of percentage of overall egg hatch rates by dose forthe 18 day egg collection period; n=1-4 replication/treatment/day; 5-478eggs/replication depending on the day and availability of eggs. The datain FIG. 3C show a box plot of relative expression of VgR at day 6 afterVgR dsRNA treatment at different doses. The data in FIG. 3C show thecorrelation of increasing dose with a larger decrease in expression ofVgR. The treatment group data were normalized to the expression foruntreated beetles.

Example 5: Gene Suppression Analysis of VgR Fragments

The effect of five distinct VgR dsRNA target fragments was assessed.FIG. 4A shows schematically the relative position of the differentfragments tested aligned against SEQ ID NO: 2. The target fragmentstested were as follows: Frag1 is VgR fragment 1 (SEQ ID NO: 3); Frag2 isVgR fragment 2 (SEQ ID NO: 4); Frag3 is VgR fragment 3 (SEQ ID NO: 5);Frag4 is VgR fragment 4 (SEQ ID NO: 6); and Frag5 is VgR fragment 5 (SEQID NO: 7). Each VgR dsRNA fragment was tested using the dietincorporation methodology described above with WCRW female beetles withthe VgR dsRNA at 100 ppm in the diet plug. The beetles were treatedindividually for one day and fed with standard diet with no dsRNA for anadditional six days. The individual beetles were then collected andflash frozen in in liquid nitrogen. For the qRTPCR assays, at least 3insects were used for each treatment group using the primer sequencesindicated in Table 3 below. FIG. 4B shows a box plot of the relative VgRexpression at day 6 after treatment with the indicated dsVgR fragmentsor control treatment (i.e., ddH2O and dsGUS (SEQ ID NO: 105, herein), asindicated, replacing the VgR dsRNA in the diet) using 5′ qRTPCR assay.The box plot shows four quartiles: average (horizontal solid line),median (horizontal dash line), and 95% confidence interval of the meanare shown. Similar results were also obtained with Mid- and 3′-qRTPCRassays. The data in the treatment groups were normalized to dataobtained from qRTPCR from untreated 3rd instar larvae.

TABLE 3 Primer Sequences Gene Suppression Analysis. Forward ReverseAmplicon qRTPCR Primer Primer Amplicon Length Assay ID SEQ ID NO. SEQ IDNO. SEQ ID NO. (nc) DV-VgR 5′ 90 91 92 147 DV-VgR mid 93 94 95 140DV-VgR 3′ 96 97 98 78 DV-RPS10 99 100 101 77

Example 6: WCRW VgR Fragment Screen by Gene Suppression Analysis

Additional VgR dsRNA fragments covering the entirety of the coding DNAsequence of SEQ ID NO: 2 were assessed for ability to suppressexpression of VgR. The fragments tested are shown aligned against SEQ IDNO: 2 in FIG. 5A, and the various sequence names correspond to thefragment ID shown in Table 1. Each VgR dsRNA fragment was tested usingthe diet incorporation methodology described above with WCRW femalebeetles with the VgR dsRNA at 100 ppm in the diet plug. The beetles weretreated individually for one day and fed with standard diet with nodsRNA for 6 additional days. The individual beetles were then collectedand flash frozen in in liquid nitrogen. For the qRTPCR assays, at least6 insects were used for each treatment group. FIGS. 5B-5D box plots ofthe relative VgR expression at day 6 after treatment with the indicateddsVgR fragments or control treatment (i.e., ddH2O and dsGUS, asindicated, replacing the VgR dsRNA in the diet) using 5′ qRTPCR assay.The data in the treatment groups were normalized to data obtained fromqRTPCR from water treated beetles. Two qRTPCR assays (5′- and Mid-qRTPCRassays) were used to avoid overlapping of VgR fragment and PCR amplicon.

Example 7: Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize with a silencingelement of the invention, the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Such as a construct can, forexample, express a long double stranded RNA of the target sequence setforth in table 1. Such a construct can be linked to a promoter. Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the polynucleotide comprising the silencing element to atleast one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos are incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos are cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.

Example 8: WCRW VgR Transgenic Feeding Bioassay

Transgenic maize plants for VgR Frag1, Frag2 and Frag3 were generatedusing the methods described herein above and used in adult feedingbioassays for gene suppression analysis in WCRW beetles as describedherein above. The expression levels of the VgR fragments in planta weredetermined in leaf samples using in vitro transcription (IVT) productsas controls. The expression analyses were carried out according tomanufacturer's instruction (Quantigene 2.0 Assay, Affymetrix, SantaClara, Calif. 95051). The average VgR fragment expression level (pg VgRfragment/mg fresh plant weight) in leaves is indicated at below eachgraph in FIGS. 6A and 6B. FIG. 6A shows data obtained from three youngplants at about the V4 growth stage for either a non-transgenic control(NTG) or the indicated transgenic planted expressing the indicatedfragment VgR Frag1 (SEQ ID NO: 3), Frag2 (SEQ ID NO: 4), and Frag3 (SEQID NO: 5). The test plants were infested with at least 14 young femalebeetles in cages. The beetles were collected at 8 days after feeding andused for gene suppression analysis. Data were normalized to expressionlevels obtained in beetles exposed NTG plants. FIG. 6B shows resultsobtained using either non-transgenic control plants or transgenic plantsexpressing the indicated VgR dsRNA fragment. The individual R1 maizeplants were infested with at least 6 young female beetles in cages.Beetles were collected 12 days after feeding for VgR expressionanalysis. Each fragment and control is represented by 2 plants used forfeeding and more than 12 insects used in gene suppression analysis, anddata were normalized to data obtained from non-transgenic control plantsexposed under similar conditions.

Example 9: WCRW Adult VgR Transgenic Exposure Bioassay

At least 32 pairs of newly emerged adult beetles were exposed to 8 daysto above ground plant part of T1 transgenic events or non-transgenic(NTG) control plants. Beetles were recollected and at least 13-37 femalebeetles were arranged in a cage for each treatment and maintained for 15days for fecundity assessment. Transgenic constructs expressed VgR Frag1(SEQ ID NO: 3), Frag2 (SEQ ID NO: 4), or Frag3 (SEQ ID NO: 5). For eachconstruct 2-4 events were tested (FIG. 7). Each cage receivedoviposition dish daily and/or at interval of 2-4 days and eggs wereprocessed following the method described in Example 2.

Example 10: WCRW Larval VgR Transgenic Exposure Bioassay

Maize T1 plants expressing silencing elements (targeting SEQ ID NOs: 3,4, and 5) were transplanted from culture plates into greenhouse flatscontaining Fafard Superfine potting mix. Three positive individualplants (of same event) were transplanted and maintained in a greenhouse(80° F., 15 hr light:9 hr darkness) and watered as needed. When theplants reached the V2 leaf stage, each pot was infested with 200non-diapausing D. virgifera virgifera eggs. Plants were monitored dailyfor first beetle emergence. The number of adult D. virgifera virgiferathat emerged from each pot was determined in the greenhouse in a similarmanner as described by Meihls et al. (2008) PNAS 105: 19177-19182. Adultbeetles emerged from T1 transgenic events and non-transgenic (NTG)control plants as in Example 9 were collected every 2 or 3 days andmaintained for 15 days for fecundity assessment. For each event threereplicate cages containing at least 8-14 pairs of male and femalebeetles were arranged. Each cage received oviposition dish every 5 daysand eggs were processed following the method described in Example 2(FIG. 8).

Example 11: WCRW Adult Exposed Sterilization Bioassay and GeneSuppression by Treatment of dsRNA Targeting BOULE

Virgin adult beetles were obtained by rearing 3^(rd) instar larvaeindividually in a 50 mL falcon tube containing the pupation medium.Beetles were sexed upon emergence; starved for 24 hours and exposed at100 ppm of dsRNA targeting the BOULE target gene (DV-BOULE-FRAG1, SEQ IDNO: 164) or controls (sterile water or GUS dsRNA) using dietincorporated method for one day. Treated beetles were provided untreateddiet and kept in solitary confinement for additional 5 days. At least 12treated beetles of mixed sex were collected in liquid nitrogen for genesuppression analysis. At least 14 pairs (male and female) were arrangedfor each treatment for subsequent mating and fecundity assessment. Dueto delay in mating, oviposition was begun 19 days after emergence andegg production was assessed for 10 days. As shown in FIG. 9A, high doseadult exposure to dsRNA targeting BOULE (BOULE dsRNA, SEQ ID NO: 164)did not affect egg production and hatch rate of the eggs. FIG. 9B showsgene expression in beetles after BOULE dsRNA (SEQ ID NO: 164) treatment.Relative expression by qRTPCR assay was performed as in previousexamples.

Example 12: WCRW Beetle Counts from Larval Exposure to T1 TransgenicPlants Expressing dsRNA Targeting BOULE

Three maize seedlings of V2 leaf stage T1 transgenic events expressingdsRNA targeting BOULE using DV-BOULE-FRAG1 (SEQ ID NO: 164) andnon-transgenic (NTG) control plants were infested with 200 WCR eggs perpot in greenhouse. Approximately one month after infestation, beetlesemerged from each pot were captured every 2 or 3 days over 10 days.Average beetle numbers for the total and each sex (Male and Female) fromat least 9 pots per event and 10 NTG control pots are shown. The boxplot shows four quartiles, average (horizontal dash line), median(horizontal solid line), and 95% confidence interval of the mean. Theresults suggest that exposure of WCRW larvae to transgenic eventsexpressing DV-BOULE FRAG1 (SEQ ID NO: 164) dsRNA did not affect adultemergence pattern when compared to NTG plants (FIG. 10). Averageexpression levels of the BOULE fragment in planta for each event weredetermined in root samples using in vitro transcription (IVT) product ascontrol as described in Example 8.

Example 13: WCRW Larval Exposure to Transgenic T1 Plants ExpressingdsRNA Targeting BOULE Caused Adult Sterilization

Beetles emerged from T1 transgenic events expressing DV-BOULE-FRAG1 (SEQID NO: 164) dsRNA and non-transgenic (NTG) control plants weremaintained for 25 days for fecundity assessment. For each event threereplicate cages containing at least 8-14 pairs of male and femalebeetles were arranged. Each cage received oviposition dish every 5 daysand eggs were processed following the method described in Example 2except that egg hatch duration was extended up to 8 days. FIG. 11A showsthe effect of larval exposure to transgenic plants expressingDV-BOULE-FRAG1 (SEQ ID NO: 164) dsRNA on the overall average eggproduction per female and average viable eggs produced per female fromemerged beetles. FIG. 11B shows the effect of larval exposure totransgenic plants expressing DV-BOULE-FRAG1 (SEQ ID NO: 164) dsRNA onhatch rate of eggs obtained from the emerged beetles. FIG. 11C indicatesthe effect of larval exposure to transgenic plants expressingDV-BOULE-FRAG1 (SEQ ID NO: 164) dsRNA on net reduction in fecundity ofemerged adult beetles relative to NTG control.

Example 14: WCRW 3^(rd) Instar Sterilization Bioassay of Exposure todsRNA Targeting MAEL, NCLB and CUL3 at 1 ppm

Over 400 3rd instar larvae were exposed to 1 ppm of the respective dsRNAsamples (DV-MAEL-FRAG1, SEQ ID NO: 46; DV-NCLB-FRAG1, SEQ ID NO: 45; andDV-CUL3-FRAG1, SEQ ID NO: 44) following the method described on Example3. Water and GUS treatment were included as experimental controls.Treated larvae were separated into four groups, each containing about 95to 100 treated larvae and placed in pupation medium for 15 days. Emergedadults were collected, counted, and transferred to their respectiveholding cages and handled as described in Example 3. After 10 days ofoviposition period the number beetles in each replicate cage wasadjusted to at least 12-16 pairs (male and female) and fecundity wasassessed for 15 days. At least 2-3 replicate cages were setup for eachtreatment. The average total number of eggs produced per female, theaverage number of viable eggs produced per female, the average egg hatchrate; average reduction in egg production and net reduction in fecundity(both relative to water control) are shown in FIG. 12.

Example 15: WCRW Sterile Gene Screening by 3^(rd) Instar SterilizationBioassay at 50 ppm

The effect of treatment of larvae on WCRW sterilization by dsRNAtargeting various genes of interest (GOI) was assessed using 3^(rd)instar. A set of 8-14 dsRNA samples including water and GUS control wastested at a time. The study was carried out using 10 day old 3rd instarlarvae that were harvested from corn mats and acclimatized on standardWCRW larval diet for 24 hours. Diet incorporation method was used forexposing larvae to 50 ppm final concentration of the respective testdsRNA samples in 2 mL per well of 6-well plate. A total of three 6-wellplates were prepared for each sample and about 312 larvae were exposedto water or 50 ppm of target dsRNA fragment (GOI) for 1 day (˜104larvae/plate; 16-18 larvae/well). After exposure, ˜10 3^(rd) instarlarvae were sampled for gene suppression analysis and the remainingtreated larvae were placed in pupation medium for 15 days. Emergedadults were collected, counted, and transferred to their respectiveholding cages and provided standard SCRW dry adult diet with a watersource (water agar) until the end of the study period (˜26 days). Beetleholding cages were kept at room temperature (usually from 22-25° C.);16:8 dark and light condition with no relative humidity control. After10 days of preoviposition period, for each treatment the number of maleand female pairs was adjusted to 16-20 pairs depending on beetle densityand each cage received oviposition dishes and eggs were collected over aperiod of for 15 days oviposition period, and processed following themethod described in Example 2. A set of three males and three femaleswere sampled 10 and 25 days after emergence for gene suppression. Table4 shows a consolidated summary of egg production, egg hatch andreduction in egg production and fecundity for active WCRW gene targets.Column 1 indicates GOI (gene of interest), column 2 and 3 indicate totalegg production/female and total viable eggs/female respectively duringthe 15 days egg production period; column 4-5 indicate cumulativeaverage egg hatch (±SEM); column 6 and 7 indicates average reduction inegg production (%) (±SEM); column 8 and 9 indicate average net reductionin fecundity (%) (±SEM). Note that values for controls (water and GUS)are cumulative average of 11 independent experiments, each run for theduration of 15 days of egg production period.

TABLE 4 WCRW sterile gene screening by 3rd instar sterilization bioassayat 50 ppm* Total No. Avg. Egg Reduction in egg Net reduction Total No.fertile hatch (%) production (%) in fecundity (%) SEQ. ID Target GeneEggs/female Eggs/female ±(SEM) ±(SEM) ±(SEM) NO: MEI 0 0 0.0 0.0 100.00.0 100.0 0.0 133 KNRL 96 0 0.5 0.3 59.6 9.4 99.6 0.1 132 TUD 7 1 15.00.8 95.5 0.8 98.6 0.2 118 CG3565 172 2 1.4 0.4 28.0 10.7 98.0 0.3 130CG17083 160 3 1.7 0.6 32.8 20.5 97.7 0.7 129 DM 96 8 8.7 2.7 73.5 6.095.7 1.0 125 CYCA 26 8 29.9 7.6 89.5 2.1 93.6 1.3 43 HIRA 63 5 7.6 1.857.5 6.5 93.1 1.1 116 Poe 41 8 16.8 2.9 74.2 7.2 91.9 2.2 113 EGG 52 2038.7 2.9 85.8 4.7 89.8 3.4 126 MR 45 21 45.9 3.5 87.6 2.2 89.4 1.8 128HANG 129 22 17.3 2.6 54.0 6.2 84.3 2.1 108 HTS 125 14 10.8 2.5 23.5 13.081.9 3.1 122 GSKT 82 26 32.3 3.4 67.7 4.8 80.6 2.9 40 ADE2 70 28 40.38.3 75.1 7.0 80.1 5.6 107 DLG1 202 39 19.3 4.8 44.3 7.1 79.9 2.6 124SU(VAR)205 132 32 24.3 4.7 52.7 9.3 77.3 4.4 111 CDK7 148 45 30.1 3.659.2 7.0 77.1 3.9 123 HRG 200 50 25.2 4.5 45.0 9.4 74.2 4.4 127 MBD-like85 17 20.4 3.4 41.2 11.9 74.0 5.3 114 11NUP44A 112 29 26.1 2.8 46.2 12.570.4 6.9 120 CASP 150 27 18.0 4.8 13.4 12.4 70.2 4.3 42 FAF 101 30 29.83.5 51.5 7.9 69.5 5.0 119 TWE 257 38 14.9 1.7 −7.6 10.3 67.4 3.1 134PORIN 105 46 44.2 4.8 62.5 4.6 67.1 4.0 110 PARK 64 31 46.3 4.0 60.1 6.466.8 5.4 112 PGLYM78 80 22 27.8 4.4 44.5 13.5 66.5 8.2 115 WTS 103 4644.7 3.6 59.5 5.9 66.4 4.9 41 PUF 89 25 28.3 3.9 40.3 8.3 64.2 5.0 117KL3 130 55 42.4 7.8 53.4 8.7 60.8 7.3 109 GUDU 211 55 26.2 3.0 16.9 6.259.6 3.0 39 CYCB 214 49 22.7 2.6 10.2 6.9 58.6 3.2 131 GEK 136 36 26.22.3 17.4 9.9 52.6 5.7 121 REPH 183 72 39.2 4.0 27.7 12.5 47.3 9.1 165ARMI 316 108 34.2 4.4 12.9 10.9 44.5 7.0 166 loqs 148 42 28.4 3.9 9.815.8 44.1 9.8 167 SCNY 165 72 43.5 3.2 31.0 5.1 39.0 4.5 168 AG03 186 4624.9 2.5 −13.7 14.0 38.1 7.6 169 DIA 288 124 43.0 4.9 20.8 9.9 36.5 7.9170 DNC 274 101 37.0 3.3 9.5 21.5 36.1 15.2 171 Chi 269 103 38.2 3.811.3 18.3 35.3 13.3 172 SXL 188 66 35.2 3.6 10.2 13.7 33.3 10.2 173 SLGA199 94 47.1 3.9 21.5 8.1 31.3 7.1 174 PAPLA1 150 53 35.4 2.7 8.7 11.629.4 8.9 175 GUS* 187 88 43.8 1.3 12.1 4.3 16.8 5.1 H20* 217 110 50.01.0 *Column 1 indicates GOI (gene of interest), column 2 and 3 indicatetotal egg production/female and total viable eggs/female respectivelyduring the 15 days egg production period; column 4-5 indicate cumulativeaverage egg hatch (±SEM); column 6 and 7 indicates average reduction inegg production (%) (±SEM); column 8 and 9 indicate average net reductionin fecundity (%) (±SEM). Note that values for controls (water and GUS)are cumulative average of 11 independent experiments, each run for theduration of 15 days of egg production period.

1. A silencing element comprising at least one double-stranded RNAregion, at least one strand of which comprises a polynucleotide that iscomplementary to: (a) the nucleotide sequence comprising any one of SEQID NOS: 1-53 or 107-253; or variants and fragments thereof, andcomplements thereof; (b) the nucleotide sequence comprising at least 90%sequence identity to any one of nucleotides SEQ ID NOS: 1-53 or 107-253;or variants and fragments thereof, and complements thereof, or (c) thenucleotide sequence comprising at least 19 consecutive nucleotides ofany one of SEQ ID NOS: 1-53 or 107-253; or variants and fragmentsthereof, and complements thereof, wherein the silencing element hassterilization activity against an insect plant pest.
 2. (canceled) 3.The silencing element of claim 1, wherein the insect plant pest is aColeoptera plant pest.
 4. The silencing element of claim 3, wherein theColeoptera plant pest is a Diabrotica plant pest.
 5. The silencingelement of claim 4, wherein the Diabrotica plant pest comprises D.virgifera virgifera, D. virgifera zeae, D. speciosa, D. barberi, D.virgifera zeae, or D. undecimpunctata howardi. 6-19. (canceled)
 20. Thesilencing element of claim 1, wherein the silencing element comprises ahairpin loop.
 21. The silencing element of claim 20, wherein thesilencing element comprises, a first segment, a second segment, and athird segment, wherein a. the first segment comprises at least about 19nucleotides having at least 90% sequence complementarity to a sequenceset forth in any one of SEQ ID NOS: 1-53 or 107-253; or variants andfragments, and complements thereof, or the first segment consists of atleast 19 nucleotides having at least 90% sequence complementarity to asequence set forth in any one of SEQ ID NOS: 1-53 or 107-253; b. thesecond segment comprises a loop of sufficient length to allow thesilencing element to be transcribed as a hairpin RNA; and, c. the thirdsegment comprises at least about 19 nucleotides having at least 85%complementarity to the first segment; wherein the second segment that isnot complementary a sequence set forth in any one of SEQ ID NOS: 1-53 or107-253, or to the other segments of the construct; and wherein thefirst and third segments form at least a partially double-strandedregion. 22-25. (canceled)
 26. The silencing element of claim 21, whereinthe first segment is complementary to a Coleoptera insect species; andwherein the third segment is complementary to a different Coleopterainsect species.
 27. (canceled)
 28. (canceled)
 29. A DNA constructcomprising a polynucleotide encoding the silencing element of claim 1.30. An expression construct comprising a DNA construct of claim
 29. 31.The expression cassette of claim 30, wherein the polynucleotide isoperably linked to a heterologous promoter.
 32. The expression cassetteof claim 30, wherein the polynucleotide is flanked by a first operablylinked convergent promoter at one terminus of the polynucleotide and asecond operably linked convergent promoter at the opposing terminus ofthe polynucleotide, wherein the first and the second convergentpromoters are capable of driving expression of the silencing element.33. A host cell comprising the DNA construct of claim
 29. 34. The hostcell of claim 33, wherein the host cell is a bacterial cell.
 35. Thehost cell of claim 34, wherein the bacterial cell is an inactivatedbacterial cell.
 36. A host cell comprising the expression construct ofclaim
 30. 37. (canceled)
 38. (canceled)
 39. A composition comprising theribonucleic acid construct of claim
 1. 40. The composition of claim 39,further comprising an agriculturally acceptable carrier.
 41. Thecomposition of claim 39, further comprising a herbicide compound, aninsecticide, a fungicide, a nematocide, an agriculturally-acceptablecarrier, and/or a bacteria, or combinations thereof.
 42. The compositionof claim 39, wherein the composition is in liquid form, solid form, orgel form.
 43. (canceled)
 44. The composition of claim 42, wherein thesolid form is a pellet, a powder, an aggregate, or a molded article. 45.A plant cell having stably incorporated into its genome a heterologouspolynucleotide encoding a silencing element, wherein the polynucleotidecomprises: a. the nucleotide sequence comprising any one of SEQ ID NOS:1-53 or 107-253; or variants and fragments thereof, and complementsthereof; b. the nucleotide sequence comprising at least 90% sequenceidentity to any one of nucleotides SEQ ID NOS: 1-53 or 107-253; orvariants and fragments thereof, and complements thereof, or c. thenucleotide sequence comprising at least 19 consecutive nucleotides ofany one of SEQ ID NOS: 1-53 or 107-253; or variants and fragmentsthereof, and complements thereof, wherein the silencing elementdecreases the fertility of an insect plant pest.
 46. (canceled)
 47. Theplant cell of claim 45, wherein the insect plant pest is a Coleopteraplant pest.
 48. The plant cell of claim 47, wherein the Coleoptera plantpest is a Diabrotica plant pest.
 49. The plant cell of claim 48, whereinthe Diabrotica plant pest comprises D. virgifera virgifera, D. virgiferazeae, D. speciosa, D. barberi, D. virgifera zeae, or D. undecimpunctatahowardi. 50.-63. (canceled)
 64. The plant cell of claim 45, wherein theplant cell further comprises an expression cassette, wherein theexpression cassette comprises the heterologous polynucleotide encoding asilencing element.
 65. The plant cell of claim 45, wherein the silencingelement expresses a double stranded RNA.
 66. The plant cell of claim 45,wherein the silencing element expresses a hairpin RNA.
 67. The plantcell of claim 45, wherein the polynucleotide is operably linked to aheterologous promoter.
 68. The plant cell of claim 45, wherein the plantcell is from a monocot.
 69. The plant cell of claim 68, wherein themonocot is maize, barley, millet, wheat or rice.
 70. The plant cell ofclaim 45, wherein the plant cell is from a dicot.
 71. The plant cell ofclaim 70, wherein the dicot is kale, cauliflower, broccoli, mustardplant, cabbage, pea, clover, alfalfa, broad bean, tomato, cassava,soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton.
 72. A plant or plant part comprising the plant cell of claim 45.73. A transgenic seed from the plant of claim
 72. 74. A method forcontrolling a plant insect pest comprising feeding to a plant insectpest a composition comprising a silencing element, wherein the silencingelement controls the plant pest, wherein the silencing element comprisesa sequence complementary to: a. the nucleotide sequence comprising anyone of SEQ ID NOS: 1-53 or 107-253; or variants and fragments thereof,and complements thereof; b. the nucleotide sequence comprising at least90% sequence identity to any one of nucleotides SEQ ID NOS: 1-53 or107-253; or variants and fragments thereof, and complements thereof, orc. the nucleotide sequence comprising at least 19 consecutivenucleotides of any one of SEQ ID NOS: 1-53 or 107-253; or variants andfragments thereof, and complements thereof, or wherein the silencingelement has insect sterilization activity against the plant pest. 75.The method of claim 74, wherein the composition comprises a plant orplant part having stably incorporated into its genome a polynucleotideencoding the silencing element.
 76. The method of claim 74, wherein thesilencing element comprises a double stranded RNA.
 77. The method ofclaim 74, wherein the silencing element comprises a hairpin RNA.
 78. Themethod of claim 75, wherein the polynucleotide encoding silencingelement is operably linked to a heterologous promoter.
 79. (canceled)80. The method of claim 74, wherein the silencing element comprises, afirst segment, a second segment, and a third segment, wherein a. thefirst segment comprises at least about 19 nucleotides having at least90% sequence complementarity to a sequence set forth in any one of SEQID NOS: 1-53 or 107-253; or variants and fragments, and complementsthereof; b. the second segment comprises a loop of sufficient length toallow the silencing element to be transcribed as a hairpin RNA; and, c.the third segment comprises at least about 19 nucleotides having atleast 85% complementarity to the first segment; wherein the secondsegment that is not complementary a sequence set forth in any one of SEQID NOS: 1-53 or 107-253; or to the other segments of the construct; andwherein the first and third segments form at least a partiallydouble-stranded region. 81.-84. (canceled)
 85. The method of claim 80,wherein the first segment is complementary to a Coleoptera insectspecies; and wherein the third segment is complementary to a differentColeoptera insect species.
 86. (canceled)
 87. (canceled)
 88. The methodof claim 74, wherein the plant is a monocot.
 89. The method of claim 88,wherein the monocot is maize, barley, millet, wheat or rice.
 90. Themethod of claim 74, wherein the plant is a dicot.
 91. The method ofclaim 90, wherein the dicot is kale, cauliflower, broccoli, mustardplant, cabbage, pea, clover, alfalfa, broad bean, tomato, cassava,soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton. 92-110. (canceled)